Methods of treating prostate cancer with anti-prostate specific membrane antigen antibodies

ABSTRACT

Modified antibodies, or antigen-binding fragments thereof, to the extracellular domain of human prostate specific membrane antigen (PSMA) are provided. The modified anti-PSMA antibodies, or antigen-binding fragments thereof, have been rendered less immunogenic compared to their unmodified counterparts to a given species, e.g., a human. Pharmaceutical compositions including the aforesaid antibodies, nucleic acids, recombinant expression vectors and host cells for making such antibodies and fragments are also disclosed. Methods of using the antibodies of the invention to detect human PSMA, or to ablate or kill a PSMA-expressing cell, e.g., a PSMA-expressing cancer or prostatic cell, either in vitro or in vivo, are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/371,399, filed Feb. 13, 2009, which is a continuation of U.S. patentapplication Ser. No. 10/449,379, filed May 30, 2003, issued as U.S. Pat.No. 7,514,078, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/379,838, filed Mar. 3, 2003, now abandoned,which is a continuation-in-part of U.S. patent application Ser. No.10/160,505, filed May 30, 2002, issued as U.S. Pat. No. 7,045,605, whichclaims priority to U.S. provisional application No. 60/295,214 filed onJun. 1, 2001, 60/323,585 filed on Sep. 20, 2001, and 60/362,810 filed onMar. 8, 2002, the contents of all of which are incorporated herein byreference.

This invention was made with government support under Department ofDefense Grant number DAMD17-98-1-8594. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates to antibodies, e.g., modified, e.g.,deimmunized, antibodies, to the extracellular domain of human prostatespecific membrane antigen (PSMA) and their uses in treating, preventing,and diagnosing prostatic disorders and cancers.

BACKGROUND OF THE INVENTION

Prostate cancer is one of the most common causes of cancer deaths inAmerican males. In 1999, approximately 185,000 new cases were diagnosedand 37,500 died of this disease (NCI SEER data). It accounts for about40% of all cancers diagnosed in men. A male born in the U.S. in 1990 hasapproximately a 1 in 8 likelihood of being diagnosed with clinicallyapparent prostate cancer in his lifetime. Even prior to the recentincrease in incidence, prostate cancer was the most prevalent cancer inmen (Feldman, A. R. et al. (1986) NEJM 315:1394-7).

There is currently very limited treatment for prostate cancer once ithas metastasized (spread beyond the prostate). Currently, systemictherapy is limited to various forms of androgen (male hormone)deprivation. While most patients will demonstrate initial clinicalimprovement, virtually inevitably, androgen-independent cells develop.Endocrine therapy is thus palliative, not curative. In a study of 1,387patients with metastatic disease detectable by imaging (e.g., bone or CTscan), the median time to objective disease progression (excludingbiochemical/PSA progression) after initiation of hormonal therapy (i.e.,development of androgen-independence) was 16-48 months (Eisenberger M.A., et al. (1998) NEJM 339:1036-42). Median overall survival in thesepatients was 28-52 months from the onset of hormonal treatment(Eisenberger M. A., et al. (1998) supra). Subsequent to developingandrogen-independence, there is no effective standard therapy and themedian duration of survival is 9-12 months (Vollmer, R. T., et al.(1999) Clin Can Res 5: 831-7; Hudes G., et al., (1997) Proc Am Soc ClinOncol 16:316a (abstract); Pienta K. J., et al. (1994) J Clin Oncol12(10):2005-12; Pienta K. J., et al. (1997) Urology 50:401-7; Tannock I.F., et al., (1996) J Clin Oncol 14:1756-65; Kantoff P. W., et al.,(1996) J. Clin. Oncol. 15 (Suppl):25:110-25). Cytotoxic chemotherapy ispoorly tolerated in this age group and generally considered ineffectiveand/or impractical. In addition, prostate cancer is relatively resistantto cytotoxic agents. Thus, chemotherapeutic regimen has not demonstrateda significant survival benefit in this patient group.

For men with a life expectancy of less than 10 years, watchful waitingis appropriate where low-grade, low-stage prostate cancer is discoveredat the time of a partial prostatectomy for benign hyperplasia (W. J.Catalona, (1994) New Engl. J. Med., 331(15):996-1004). Such cancersrarely progress during the first five years after detection. On theother hand, for younger men, curative treatment is often moreappropriate.

Where prostate cancer is localized and the patient's life expectancy is10 years or more, radical prostatectomy offers the best chance foreradication of the disease. Historically, the drawback of this procedureis that most cancers had spread beyond the bounds of the operation bythe time they were detected. However, the use of prostate-specificantigen testing has permitted early detection of prostate cancer. As aresult, surgery is less extensive with fewer complications. Patientswith bulky, high-grade tumors are less likely to be successfully treatedby radical prostatectomy.

After surgery, if there are detectable serum prostate-specific antigenconcentrations, persistent cancer is indicated. In many cases,prostate-specific antigen concentrations can be reduced by radiationtreatment. However, this concentration often increases again within twoyears.

Radiation therapy has also been widely used as an alternative to radicalprostatectomy. Patients generally treated by radiation therapy are thosewho are older and less healthy and those with higher-grade, moreclinically advanced tumors. Particularly preferred procedures areexternal-beam therapy which involves three dimensional, conformalradiation therapy where the field of radiation is designed to conform tothe volume of tissue treated; interstitial-radiation therapy where seedsof radioactive compounds are implanted using ultrasound guidance; and acombination of external-beam therapy and interstitial-radiation therapy.

For treatment of patients with locally advanced disease, hormonaltherapy before or following radical prostatectomy or radiation therapyhas been utilized. Hormonal therapy is the main form of treating menwith disseminated prostate cancer. Orchiectomy reduces serumtestosterone concentrations, while estrogen treatment is similarlybeneficial. Diethylstilbestrol from estrogen is another useful hormonaltherapy which has a disadvantage of causing cardiovascular toxicity.When either LHRH agonists, such as leuprolide, buserelin, or goserelin,or gonadotropin-releasing hormone antagonists, such as Abarclix, areadministered testosterone concentrations are ultimately reduced.Flutamide and other nonsteroidal, anti-androgen agents block binding oftestosterone to its intracellular receptors. As a result, it blocks theeffect of testosterone, increasing serum testosterone concentrations andallows patients to remain potent—a significant problem after radicalprostatectomy and radiation treatments.

In view of the shortcoming of existing therapies, there exists a needfor improved modalities for preventing and treating cancers, such asprostate cancer.

SUMMARY OF THE INVENTION

This invention provides, inter alia, antibodies and particularly,modified antibodies, or antigen-binding fragments thereof, that bind tothe extracellular domain of human prostate specific membrane antigen(PSMA). The modified anti-PSMA antibodies, or antigen-binding fragmentsthereof, have been rendered less immunogenic compared to theirunmodified counterparts to a given species, e.g., a human. The modifiedanti-PSMA antibodies, or fragments thereof, bind to human PSMA with highaffinity and specificity, and thus can be used as diagnostic,prophylactic, or therapeutic agents in vivo and in vitro. Accordingly,the invention provides antibodies and particularly modified anti-PSMAantibodies, antibody fragments, and pharmaceutical compositions thereof,as well as nucleic acids, recombinant expression vectors and host cellsfor making such antibodies and fragments. Methods of using theantibodies of the invention to detect PSMA, or to ablate or kill aPSMA-expressing cell, e.g., a PSMA-expressing cancer, a prostatic, or avascular cell, either in vitro or in vivo, are also encompassed by theinvention. Preferably, the modified antibodies are those having one ormore complementarity determining regions (CDRs) from a J591, J415, J533or E99 antibody. As discussed herein, the modified antibodies can beCDR-grafted, humanized, deimmunized, or, more generally, antibodieshaving the CDRs from a non-human antibody, e.g., murine J591, J415, J533or E99 antibody, and a framework that is selected as less immunogenic inhumans, e.g., less antigenic than the murine frameworks in which amurine CDR naturally occurs.

The antibodies, e.g., modified antibodies of the invention interactwith, e.g., bind to, PSMA, preferably human PSMA, with high affinity andspecificity. For example, the antibody binds to human PSMA with anaffinity constant of at least 10⁷M⁻¹, preferably between 10⁸M⁻¹ and 10¹⁰M⁻¹, or about 10⁹M⁻¹. Preferably, the antibody interacts with, e.g.,binds to, the extracellular domain of PSMA, and most preferably, theextracellular domain of human PSMA (e.g., amino acids 44-750 of humanPSMA).

In some embodiments, the anti-PSMA antibody binds all or part of anepitope bound by an antibody described herein, e.g., a J591, E99, J415,and J533 antibody. The anti-PSMA antibody can inhibit, e.g.,competitively inhibit, the binding of an antibody described herein,e.g., a J591, E99, J415, and J533 antibody, to human PSMA. An anti-PSMAantibody may bind to an epitope, e.g., a conformational or a linearepitope, which epitope when bound prevents binding of an antibodydescribed herein, e.g., a J591, E99, J415, and J533 antibody. Theepitope can be in close proximity spatially or functionally-associated,e.g., an overlapping or adjacent epitope in linear sequence orconformational space, to the one recognized by the J591, E99, J415, orJ533 antibody.

In some embodiments, the anti-PSMA antibody binds to an epitope locatedwholly or partially within the region of about amino acids 120 to 500,preferably 130 to 450, more preferably, 134 to 437, or 153 to 347, ofhuman PSMA. Preferably, the epitope includes at least one glycosylationsite, e.g., at least one N-linked glycosylation site (e.g., theasparagine residue located at about amino acids 190-200, preferably atabout amino acid 195, of human PSMA).

Human PSMA is expressed on the surface of normal, benign hyperplastic,and cancerous prostate epithelial cells, as well as vascular endothelialcells proximate to cancerous cells, e.g., renal, urothelial (e.g.,bladder), testicular, colon, rectal, lung (e.g., non-small cell lungcarcinoma), breast, liver, neural (e.g., neuroendocrine), glial (e.g.,glioblastoma), pancreatic (e.g., pancreatic duct), melanoma (e.g.,malignant melanoma), or soft tissue sarcoma cancerous cells. Theexpression of human PSMA is substantially lower on non-malignantprostate cells where PSM′, a splice variant that lacks a portion of theN-terminal domain that includes the transmembrane domain, is moreabundant. Due to the absence of the N-terminal region containing thetransmembrane domain, PSM′ is primarily cytoplasmic and is not locatedon the cell membrane. The antibodies, e.g., the modified antibodies, ofthe invention bind to the cell surface of cells that express PSMA. PSMAis normally recycled from the cell membrane into the cell. Thus, theantibodies of the invention are internalized with PSMA through theprocess of PSMA recirculation, thereby permitting delivery of an agentconjugated to the antibody, e.g., a labeling agent, a cytotoxic agent,or a viral particle (e.g., a viral particle containing genes that encodecytotoxic agents, e.g., apoptosis-promoting factors). Accordingly,antibodies, e.g., modified antibodies, described herein, can be used totarget living normal, benign hyperplastic, and cancerous prostateepithelial cells, as well as vascular endothelial cells proximate tocancerous cells.

An antibody, e.g., a modified antibody, is preferably monospecific,e.g., a monoclonal antibody, or an antigen-binding fragment thereof. Theantibodies, e.g., the modified antibodies, can be full-length (e.g., anIgG (e.g., an IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2), IgD,and IgE, but preferably an IgG) or can include only an antigen-bindingfragment (e.g., a Fab, F(ab′)₂ or scFv fragment, or one or more CDRs).An antibody, or antigen-binding fragment thereof, can include two heavychain immunoglobulins and two light chain immunoglobulins, or can be asingle chain antibody. The antibodies can, optionally, include aconstant region chosen from a kappa, lambda, alpha, gamma, delta,epsilon or a mu constant region gene. A preferred anti-PSMA antibodyincludes a heavy and light chain constant region substantially from ahuman antibody, e.g., a human IgG1 constant region, a portion thereof,or a consensus sequence.

In a preferred embodiment, the antibodies (or fragments thereof) arerecombinant or modified anti-PSMA antibodies chosen from, e.g., achimeric, a humanized, a deimmunized, or an in vitro generated antibody.In other embodiments, the anti-PSMA antibodies are human antibodies. Inone embodiment, a modified antibody of the invention is a deimmunizedanti-PSMA antibody, e.g., a deimmunized form of E99, J415, J533 or J591(e.g., a deimmunized form of an antibody produced by a hybridoma cellline having an ATCC Accession Number HB-12101, HB-12109, HB-12127, andHB-12126, respectively). Preferably, a modified antibody is adeimmunized form of J591 or J415 (referred to herein as “deJ591” or“deJ415”, respectively). Most preferably, the antibody is a deimmunizedform of J591.

Any combination of anti-PSMA antibodies is within the scope of theinvention, e.g., two or more antibodies that bind to different regionsof PSMA, e.g., antibodies that bind to two different epitopes on theextracellular domain of PSMA.

In some embodiments, the anti-PSMA antibody, e.g., the modifiedanti-PSMA antibody or antigen-binding fragment thereof, includes atleast one light or heavy chain immunoglobulin (or preferably, at leastone light chain immunoglobulin and at least one heavy chainimmunoglobulin). Preferably, each immunoglobulin includes a light or aheavy chain variable region having at least one, two and, preferably,three CDRs substantially identical to a CDR from a non-human anti-PSMAlight or heavy chain variable region, respectively. For example, theantibody or antigen-binding fragment thereof can have at least one, twoand preferably three CDRs from: the heavy chain variable region ofmurine J591 (see SEQ ID NO:1, 2, and 3, depicted in FIG. 1A); the lightchain variable region of murine J591 (see SEQ ID NO:4, 5, and 6,depicted in FIG. 1B); the heavy chain variable region of murine J415(see SEQ ID NO:29, 30, and 31, depicted in FIG. 5); the light chainvariable region of murine J415 (see SEQ ID NO:32, 33, and 34, depictedin FIG. 6); the heavy chain variable region of murine J533 (see SEQ IDNO:93, 94, and 95, depicted in FIG. 9A); the light chain variable regionof murine J533 (see SEQ ID NO:96, 97, and 98, depicted in FIG. 10A); theheavy chain variable region of murine E99 (see SEQ ID NO:99, 100, and101, depicted in FIG. 11A); or the light chain variable region of murineE99 (see SEQ ID NO:102, 103, and 104, depicted in FIG. 12A). In otherembodiments, the modified antibody or antigen-binding fragment thereofcan have at least one, two, and preferably three CDRs from the light orheavy chain variable region of the antibody produced by the cell linehaving ATCC Accession Number HB-12126 or the deimmunized J591 (deJ591)antibody produced by the cell line having ATCC Accession NumberPTA-3709. In other embodiments, the modified antibody or antigen-bindingfragment thereof can have at least one, two and preferably three CDRsfrom the light or heavy chain variable region of the antibody producedby the cell line having ATCC Accession Number HB-12109 or thedeimmunized J415 antibody produced by a cell line having ATCC AccessionNumber PTA-4174. In still other embodiments, the modified antibody orantigen-binding fragment thereof can have at least one, two andpreferably three CDRs from the light or heavy chain variable region ofthe antibody produced by the cell line having ATCC Accession NumberHB-12127 or the antibody produced by a cell line having ATCC AccessionNumber HB-12101.

In one preferred embodiment, the modified antibody or antigen-bindingfragment thereof includes all six CDRs from the same non-human anti-PSMAantibody, e.g., a murine J591, J415, J533 or E99 antibody. In someembodiments, the CDRs have the amino acid sequences of SEQ ID NO:1, 2,3, 4, 5 and 6 (corresponding to murine J591 heavy and light chain CDRs),the amino acid sequences of the CDRs of the antibody produced by thecell line having ATCC Accession number HB-12126, or the deimmunized J591antibody produced by the cell line having ATCC Accession NumberPTA-3709, or sequences substantially identical thereto. In otherembodiments, the CDRs have the amino acid sequences of SEQ ID NO:29, 30,31, 32, 33, and 34 (corresponding to murine J415 heavy and light chainCDRs), the amino acid sequences of the CDRs of the antibody produced bythe cell line having ATCC Accession Number HB-12109, or the deimmunizedJ415 antibody produced by the cell line having ATCC Accession NumberPTA-4174, or sequences substantially identical thereto. In otherembodiments, the CDRs have the amino acid sequences of SEQ ID NO:93, 94,95, 96, 97, and 98 (corresponding to murine J533 heavy and light chainCDRs), the amino acid sequences of the CDRs of the antibody produced bythe cell line having ATCC Accession Number HB-12127, or sequencessubstantially identical thereto. In still other embodiments, the CDRshave the amino acid sequences of SEQ ID NO:99, 100, 101, 102, 103, and104 (corresponding to murine E99 heavy and light chain CDRs), the aminoacid sequences of the CDRs of the antibody produced by the cell linehaving ATCC Accession Number HB-12101, or sequences substantiallyidentical thereto.

The amino acid sequence of the CDRs for antibodies J591, J415, J533 andE99 are provided below in Table 1.

TABLE 1 CDR Sequences SEQ ID NAME Organism FIG. NO: SEQUENCE V_(H) CDR1Mus musculus FIG. 1A   1 GYTFTEYTIH J591 V_(H) CDR2 Mus musculus FIG. 1A  2 NINPNNGGTTYNQKFED J591 V_(H) CDR3 Mus musculus FIG. 1A   3 GWNFDYJ591 V_(L) CDR1 Mus musculus FIG. 1B   4 KASQDVGTAVD J591 V_(L) CDR2Mus musculus FIG. 1B   5 WASTRHT J591 V_(L) CDR3 Mus musculus FIG. 1B  6 QQYNSYPLT J591 V_(H) CDR1 Mus musculus FIG. 5  29 GFTFSNYWMN J415V_(H) CDR2 Mus musculus FIG. 5  30 EIRSQSNNFATHIYAESVKG J415 V_(H) CDR3Mus musculus FIG. 5  31 RWNNF J415 V_(L) CDR1 Mus musculus FIG. 6  32KASENVGTYVS J415 V_(L) CDR2 Mus musculus FIG. 6  33 GASNRFT J415V_(L) CDR3 Mus musculus FIG. 6  34 GQSYTFPYT J415 V_(H) CDR1Mus musculus FIG. 9A  93 GYTFTGYVMH J533 V_(H) CDR2 Mus musculus FIG. 9A 94 YINPYNDVTRYNGKFKG J533 V_(H) CDR3 Mus musculus FIG. 9A  95 GENWYYFDSJ533 V_(L) CDR1 Mus musculus FIG. 10A  96 RASESIDSYDNTFIVIII J533V_(L) CDR2 Mus musculus FIG. 10A  97 RASILES J533 V_(L) CDR3Mus musculus FIG. 10A  98 HQSIEDPYT J533 V_(H) CDR1 Mus musculusFIG. 11A  99 GFSLTAYGIN E99 V_(H) CDR2 Mus musculus FIG. 11A 100VIWPDGNTDYNSTLKS E99 V_(H) CDR3 Mus musculus FIG. 11A 101 DSYGNFKRGWFDFE99 V_(L) CDR1 Mus musculus FIG. 12A 102 KASQNVGSDVA E99 V_(L) CDR2Mus musculus FIG. 12A 103 STSYRYS E99 V_(L) CDR3 Mus musculus FIG. 12A104 QQYNSYPLT E99

The light or heavy chain immunoglobulin of the modified anti-PSMAantibody or antigen-binding fragment thereof can further include a lightchain or a heavy chain variable framework sequence from a light chain orheavy chain variable framework present in a human or a non-human, e.g.,rodent, antibody (e.g., the murine J591, J415, J533 or E99 antibodyheavy chain or light chain variable framework). In some embodiments, thelight chain or the heavy chain variable framework can be chosen from:

-   -   i. a light or heavy chain variable framework including at least        5, 10, 20, 30, 40, 50, 60, 70, or 80 amino acid residues from a        human light or heavy chain variable framework, e.g., a light or        heavy chain variable framework residue from a mature human        antibody, a human germline antibody sequence, or a human        consensus antibody sequence;    -   ii. a light or heavy chain variable framework including at least        5, but less than 30, amino acid residues from a human light        chain or heavy chain variable framework, e.g., a light chain or        heavy chain variable framework residue from a mature human        antibody, a human germline antibody sequence, or a human        consensus antibody sequence;    -   a light or heavy chain variable framework including at least 5,        10, 20, 30, 40, 50, 60, 75 or more amino acid residues from a        light or heavy variable framework from a non-human antibody,        e.g., a murine antibody (e.g., an anti-PSMA antibody having the        framework amino acid sequence shown in SEQ ID NO:7 or 8 (from        the heavy and light chain, respectively, of murine J591; see        FIGS. 1A and 1B), SEQ ID NO:35 or 36 (from the heavy and light        chain, respectively, of murine J415; see FIGS. 5 and 6), SEQ ID        NO:109 or 114 (from the heavy and light chain, respectively, of        murine J533; see FIGS. 9A and 10A), or SEQ ID NO: 119 or 124        (from the heavy and light chain, respectively, of murine E99;        see FIGS. 11A and 12A), or the framework of a murine antibody        described herein (e.g., a murine J591, J415, J533, or E99        antibody produced by a hybridoma cell line having an ATCC        Accession Number HB-12126, HB-12109, HB-12127 or HB-12101);    -   iv. a light or heavy chain variable framework, which has at        least 60%, 65%, 70%, 72%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,        98%, 99% or more identity with, or which has an amino acid        sequence which differs by at least 1, 2, 5, or more residues,        but less than 10, 20, 30 or 40 residues from, the sequence of        the framework of a light or heavy chain variable region of a        non-human antibody, e.g., a murine antibody (e.g., an anti-PSMA        antibody having the framework amino acid sequence shown in SEQ        ID NO:7 or 8 (from the heavy and light chain, respectively, of        murine J591; see FIGS. 1A and 1B), SEQ ID NO:35 or 36 (from the        heavy and light chain, respectively, of murine J415; see FIGS. 5        and 6), SEQ ID NO:109 or 114 (from the heavy and light chain,        respectively, of murine J533; see FIGS. 9A and 10A), or SEQ ID        NO:119 or 124 (from the heavy and light chain, respectively, of        murine E99; see FIGS. 11A and 12A)), or the framework of a        murine antibody described herein (e.g., a murine antibody        produced by a hybridoma cell line having an ATCC Accession        Number HB-12126, HB-12109, HB-12127 or HB-12101); or    -   v. a non-human, e.g., a murine, e.g., a J591 or J415, light or        heavy chain variable region framework which has at least 5 amino        acid replacements.

In some embodiments, the light chain variable region of the non-humananti-PSMA antibody or antigen-binding fragment thereof has at least one,two, three and preferably four amino acid sequences chosen from SEQ IDNO: 13, 14, 15, and 16 (corresponding to deimmunized J591 light chainFR's 1-4; see FIG. 2B) or SEQ ID NO:41, 42, 43, and 44 (corresponding todeimmunized J415 light chain (J415DIVK5) FR's 1-4; see FIG. 6), or atleast one, two, three and preferably four light chain framework regionsfrom the antibody produced by the cell line having ATCC Accession NumberPTA-3709 or PTA-4174. In other embodiments, the heavy chain variableregion of the non-human anti-PSMA antibody or antigen binding portionthereof has at least one, two, three, and preferably four amino acidsequences chosen from SEQ ID NO:9, 10, 11, and 12 (corresponding todeimmunized J591 heavy chain FR's 1-4; see FIG. 2A) or SEQ ID NO:37, 38,39, and 40 (corresponding to deimmunized J415 heavy chain (J415DIVH4)FR's 1-4; see FIG. 5), or at least one, two, three and preferably fourheavy chain framework regions of the antibody produced by the cell linehaving ATCC Accession Number PTA-3709 or PTA-4174. In other embodiments,the heavy or light chain framework has an amino acid sequence which hasat least 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identity with SEQ IDNO:17 or SEQ ID NO:18, respectively (corresponding to deimmunized J591framework sequence; see FIGS. 2A-2B), SEQ ID NO:45 or SEQ ID NO:46,respectively (corresponding to deimmunized J415 framework sequencesJ415DIVH4 and J415DIVK5; see FIG. 5 or 6), or with the heavy or lightchain framework sequence of an antibody produced by the cell line havingATCC Accession Number PTA-3709 or PTA-4174. In still other embodiments,the heavy or light chain framework has an amino acid sequence whichdiffers by at least 1, 2, 5, or more residues, but less than 10, 20, 30,or 40 residues, from the amino acid sequence of SEQ ID NO:17 or SEQ IDNO:18, respectively, SEQ ID NO:45 or SEQ ID NO:46, respectively, or theheavy or light chain framework sequence of the antibody produced by thecell line having ATCC Accession Number PTA-3709 or PTA-4174. Preferably,the heavy or light chain framework region includes the amino acidsequence shown in SEQ ID NO:17 or SEQ ID NO:18, respectively, SEQ IDNO:45 or SEQ ID NO:46, respectively, or the heavy or light chainframework sequence of the antibody produced by the cell line having ATCCAccession Number PTA-3709 or PTA-4174.

In other embodiments, the heavy or light chain variable region of themodified anti-PSMA antibody has an amino acid sequence which has atleast 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identity with SEQ IDNO:21 or SEQ ID NO:22, respectively (corresponding to the heavy andlight chain variable regions of deimmunized J591; see FIGS. 2A-2B), SEQID NO:49 or SEQ ID NO:50, respectively (corresponding to the heavy andlight chain variable regions of deimmunized J415, J415DIVH4 andJ415DIVK5; see FIG. 5 or 6), or the heavy or light chain variable regionsequence of the antibody produced by the cell line having ATCC AccessionNumber PTA-3709 or PTA-4174. In other embodiments, the heavy or lightchain variable region of the modified anti-PSMA antibody has an aminoacid sequence that differs by at least 1, 2, 5, or more residues, butless than 10, 20, 30, or 40 residues, from the amino acid sequence ofSEQ ID NO:21 or SEQ ID NO:22, respectively, SEQ ID NO:49 or SEQ IDNO:50, respectively, or the heavy or light chain variable regionsequence of the antibody produced by the cell line having ATCC AccessionNumber PTA-3709 or PTA-4174. Preferably, the light or heavy chainvariable region includes the amino acid sequence shown in SEQ ID NO:21or SEQ ID NO:22, respectively, SEQ ID NO:49 or SEQ ID NO:50,respectively, or the heavy or light chain variable region sequence ofthe antibody produced by the cell line having ATCC Accession NumberPTA-3709 or PTA-4174.

Preferred modified anti-PSMA antibodies include at least one, preferablytwo, light chain variable regions and at least one, preferably two,heavy chain variable regions having the amino acid sequence shown in SEQID NO:21 and SEQ ID NO:22, respectively (corresponding to the heavy andlight chain variable regions of deimmunized J591; see FIGS. 2A-2B), SEQID NO:49 and SEQ ID NO:50, respectively (corresponding to the heavy andlight chain variable regions of deimmunized J415, J415DIVH4 andJ415DIVK5; see FIGS. 5 and 6), or at least one, preferably two, modifiedlight chain variable region sequences and at least one, preferably two,heavy chain variable region sequences of the antibody produced by thecell line having ATCC Accession Number PTA-3709 or PTA-4174.

In other embodiments, the light or heavy chain variable framework of theanti-PSMA antibody, or antigen-binding fragment thereof, includes atleast one, two, three, four, five, six, seven, eight, nine, ten,fifteen, sixteen, or seventeen amino acid residues from a human light orheavy chain variable framework, e.g., a light or heavy chain variableframework residue from a mature human antibody, a human germlineantibody sequence, or a consensus antibody sequence.

In some embodiments, the amino acid residue from the human light chainvariable framework is the same as the residue found at the same positionin a human germline antibody sequence. Preferably, the amino acidresidue from the human light chain variable framework is the most commonresidue at the same position in the human germline antibody sequence.Preferably, the light chain variable framework of the modified anti-PSMAantibody, or antigen-binding fragment thereof, has at least one, two,three, five, seven, ten amino acid residues which differ from theframework of the non-human anti-PSMA light chain variable region (e.g.,the murine J591 light chain variable region), or which is from a humanlight chain variable framework (e.g., a human germline, mature, orconsensus framework sequence), at a position selected from the groupconsisting of: residue 8, 9, 10, 11, 20, 22, 60, 63, 76, 77, 78, 80, 83,87, 103, 104 and 106 (Kabat numbering as shown in Table 2). Preferably,the light chain variable framework of the modified anti-PSMA antibody,or antigen-binding fragment thereof, has at least one, two, three, five,seven, or ten amino acid residues from the human light chain variableframework selected from the group consisting of: residue 8 (proline), 9(serine), 10 (serine), 11 (leucine), 20 (threonine), 22 (threonine), 60(serine), 63 (serine), 76 (serine), 77 (serine), 78 (leucine), 80(proline), 83 (phenylalanine), 87 (tyrosine), 103 (lysine), 104 (valine)and 106 (isoleucine) (Kabat numbering as shown in Table 2).

The amino acid replacements in the deimmunized J591 light chain variableregion are provided below in Table 2. The left panel indicates the aminoacid number according to Kabat, E. A., et al. (1991) supra; the middlepanel indicates the replacements of the residue in the mouse sequenceand the corresponding mouse residues; and the right panel indicates themost common residue in the corresponding position in the human germline.

TABLE 2 Position Substitution Most common in Kabat No of mouse sequencehuman germline 3 V→Q V 8 H→P P 9 K→S S 10 F→S S 11 M→L L 20 S→T T 21 I→LI 22 I→T T 42 Q→P K 58 V→I V 60 D→S S 63 T→S S 76 T→S S 77 T→S S 78 V→LL 80 S→P P 83 L→F F 87 F→Y Y 100 A→P Q 103 M→K K 104 L→V V 106 L→I I

In other embodiments, the light chain variable framework of theanti-PSMA antibody, or antigen-binding fragment thereof, has at leastone, two, three, five, or seven amino acid residues which differ fromthe framework of a non-human anti-PSMA light chain variable region(e.g., the murine J415 light chain variable region), or which is from ahuman light chain variable framework (e.g., a human germline, mature, orconsensus framework), at a position selected from the group consistingof: residue 13, 15, 19, 41, 63, 68, and 80 (linear numbering as shown inFIG. 6). Preferably, the light chain variable framework of the modifiedantibody, or antigen-binding fragment thereof, has at least one, two,three, five, or seven amino acid residues from the human consensus lightchain variable framework selected from the group consisting of: residue13 (alanine), 15 (alanine), 19 (methionine), 41 (threonine), 63(serine), 68 (glycine), and 80 (alanine) (linear numbering as shown inFIG. 6).

The amino acid replacements in the deimmunized J415 light chain variableregion are provided below in Table 3. The left panel indicates the aminoacid number using linear numbering; the middle panel indicates thereplacements of the residue in the mouse sequence and the correspondingmouse residues; and the right panel indicates the most common residue inthe corresponding position in the human germline.

TABLE 3 Position Substitution Most common in Linear No of mouse sequencehuman germline 13 I→A A 15 V→A A 19 V→M M 41 E→T T 63 T→S S 68 A→G G 80T→A A

In other embodiments, the light chain variable framework of theanti-PSMA antibody, or antigen-binding fragment thereof, includes atleast 5, but no more than 80, amino acid residues from the light chainvariable framework shown in SEQ ID NO:8 (from murine J591; see FIG. 1B),SEQ ID NO:36 (from murine J415; see FIG. 6), SEQ ID NO: 114 (from murineJ533; see FIG. 10A), or SEQ ID NO:124 (from murine E99; see FIG. 12A),or the light chain variable framework of an antibody produced by thehybridoma cell line having an ATCC Accession Number HB-12126, HB-12109,HB-12127 or HB-12101. Preferably, the light chain variable framework hasat least 60%, 65%, 70%, 72%, 75%, 80%, 85%, 90%, or 94% identity with,or differs by at least 5, 7, 10, 20, or 30 but less than 10, 20, 30, or40 amino acid residues from, the non-human light chain variableframework, e.g., the murine J591 or J415 light chain variable frameworkshown in SEQ ID NO:8 or SEQ ID NO:36, respectively, or the light chainvariable framework of the antibody produced by the hybridoma cell linehaving an ATCC Accession Number HB-12126 or HB-12109. In otherembodiments, the light chain variable framework is from murine J591antibody (SEQ ID NO:8; see FIG. 1B), from murine J415 antibody (SEQ IDNO:36; see FIG. 6), from murine J533 antibody (SEQ ID NO:114; see FIG.10A), or from murine E99 antibody (SEQ ID NO:124; see FIG. 12A), or thelight chain variable framework of the antibody produced by the hybridomacell line having an ATCC Accession Number HB-12126, HB-12109, HB-12127or HB-12101.

In yet other embodiments, the light chain variable framework of themodified anti-PSMA antibody, or antigen-binding fragment thereof,includes a non-human (e.g., a murine) light chain variable framework(e.g., a murine J591 light chain variable framework as shown in SEQ IDNO:8 or the light chain variable framework of the antibody produced bythe hybridoma cell line having an ATCC Accession Number HB-12126) whichhas at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acidreplacements. In one embodiment, the non-human light chain variableframework includes one or more of:

-   -   a framework region 1 having at least 5, 6, 7, or 8 replacements;    -   a framework region 2 having at least one replacement;    -   a framework region 3 having at least 5, 6, 7, 8, or 9        replacements; or    -   a framework region 4 having at least 2, 3 or 4 replacements.

In yet other embodiments, the light chain variable framework of themodified anti-PSMA antibody, or antigen-binding fragment thereof,includes a non-human (e.g., a murine) light chain variable framework(e.g., a murine J415 light chain variable framework as shown in SEQ IDNO:36 or the light chain variable framework of the antibody produced bythe hybridoma cell line having an ATCC Accession Number HB-12109) whichhas at least 1, 2, 3, 4, 5, 6, 7, 8, or 10 amino acid replacements. Insome embodiments, the non-human light chain variable framework includesone or more of:

-   -   a framework region 1 having at least 1, 2 or 3 replacements;    -   a framework region 2 having at least one replacement; or    -   a framework region 3 having at least 1, 2 or 3 replacements.

The replacement can be selected from: a conservative substitution of anon-human residue, or a residue found in a human germline, mature orconsensus framework sequence at the same position, e.g. the most commonresidue in the human germline sequence at the same position. In someembodiments, the light chain variable framework has at least 3, 4 andpreferably 5 conservative substitutions. In other embodiments, the lightchain variable framework has at least 5, 7, 10, 15, 16, or 17 amino acidreplacements wherein the replacement amino acid residue is the mostcommon residue in the human germline framework sequence at the sameposition.

In some embodiments, the non-human light chain variable framework has atleast one, two, three, five, seven, ten, eleven, fifteen, sixteen,seventeen, nineteen, twenty, twenty-one or twenty-two amino acidreplacements at a position selected from the group consisting of:residue 3, 8, 9, 10, 11, 20, 21, 22, 42, 58, 60, 63, 76, 77, 78, 80, 83,87, 100, 103, 104 and 106 (Kabat numbering as shown in Table 2). Thereplacement can be chosen from one or more of: residue 3 (glutamine), 8(proline), 9 (serine), 10 (serine), 11 (leucine), 20 (threonine), 21(leucine), 22 (threonine), 42 (proline), 58 (isoleucine), 60 (serine),63 (serine), 76 (serine), 77 (serine), 78 (leucine), 80 (proline), 83(phenylalanine), 87 (tyrosine), 100 (proline), 103 (lysine), 104(valine) and 106 (isoleucine) (Kabat numbering as shown in Table 2).

In other embodiments, the non-human light chain variable framework hasat least one, two, three, five, or seven amino acid replacements at aposition selected from the group consisting of: residue 13, 15, 19, 41,63, 68 and 80 (linear numbering as shown in Table 3). Preferably, thelight chain variable framework of the modified antibody, orantigen-binding fragment thereof, has at least one, two, three, five,seven amino acid residues from the human consensus light chain variableframework selected from the group consisting of: residue 13 (alanine),15 (alanine), 19 (methionine), 41 (threonine), 63 (serine), 68 (glycine)and 80 (alanine) (linear numbering as shown in Table 3).

Preferably, the heavy chain variable framework of the modified anti-PSMAantibody, or antigen-binding fragment thereof, has at least one, two,three, five, seven, or eight amino acid residues, which differ from theframework of the non-human anti-PSMA heavy chain variable region (e.g.,the murine J591 heavy chain variable region), or which is from a humanheavy chain variable framework (e.g., a human germline framework), at aposition selected from the group consisting of: residue 5, 40, 41, 44,82a, 83, 87, and 108 (Kabat numbering as shown in Table 4). Preferably,the heavy chain variable framework of the recombinant antibody, orantigen-binding fragment thereof, has at least one amino acid residuefrom the human heavy chain variable framework selected from the groupconsisting of: residue 5 (valine), 40 (alanine), 41 (proline), 44(glycine), 82a (serine), 83 (arginine), 87 (threonine), or 108 (leucine)(Kabat numbering as shown in Table 4).

The amino acid replacements in the deimmunized J591 heavy chain variableregion are provided below in Table 4. The left panel indicates the aminoacid number according to Kabat, E. A., et al. (1991) supra; the middlepanel indicates the replacements of the residue in the mouse sequenceand the corresponding mouse residues; and the right panel indicates themost common residue in the corresponding position in the human germline.

TABLE 4 Position Substitution Most common in Kabat No. of mouse sequencehuman germline  5 Q→V V 11 L→V L 12 V→K V 16 T→A G 17 S→T S 19 R→K R 40S→A A 41 H→P P 44 S→G G 75 S→T K 76 S→D N   82a R→S S 83 T→R R 87 S→T T108  T→L L

In other embodiments, the heavy chain variable framework of the modifiedanti-PSMA antibody, or antigen-binding fragment thereof, has at leastone, two, three, four, five amino acid residues, which differ from theframework of a non-human anti-PSMA heavy chain variable region (e.g.,the murine J415 heavy chain variable region), or which is from a humanheavy chain variable framework (e.g., a human mature, consensus, orgermline framework), at a position selected from the group consistingof: residue 20, 87, 94, 95, and 112 (linear numbering as shown in Table5). Preferably, the heavy chain variable framework of the recombinantantibody, or antigen-binding fragment thereof, has at least one, two,three, four, five amino acid residues from the human heavy chainvariable framework selected from the group consisting of: residue 20(isoleucine), 87 (serine), 94 (alanine), 95 (valine), and 112 (valine)(linear numbering as shown in Table 5).

The amino acid replacements in the deimmunized J415 heavy chain variableregion are provided below in Table 5. The left panel indicates thelinear amino acid number; the middle panel indicates the replacements ofthe residue in the mouse sequence and the corresponding mouse residues;and the right panel indicates the most common residue in thecorresponding position in the human germline.

TABLE 5 Position Substitution Most common in Kabat No of mouse sequencehuman germline 20 L→I I 87 N→S S 94 G→A A 95 I→V V 112 L→V V

In other embodiments, the heavy chain variable framework of the modifiedanti-PSMA antibody, or antigen-binding fragment thereof, includes atleast 5 but no more than 75 or 82 amino acid residues from the heavychain variable framework shown in SEQ ID NO:7 (from murine J591; seeFIG. 1A), SEQ ID NO:35 (from murine J415; see FIG. 5), SEQ ID NO:109(from murine J533; see FIG. 9A), or SEQ ID NO:119 (from murine E99; seeFIG. 11A), or the heavy chain variable framework of the antibodyproduced by the hybridoma cell line having an ATCC Accession NumberHB-12126, HB-12109, HB-12127 or HB-12101. Preferably, the heavy chainvariable framework has at least 60%, 65%, 70%, 80%, 82%, 85%, 90%, or94% identity with, or differs by at least 5, 10, 20, or 30 but less than10, 20, 30, or 40 residues from, a non-human heavy chain variableframework, e.g., the murine J591 or J415 or heavy chain variableframework shown in SEQ ID NO:7 or SEQ ID NO:35, respectively, or a heavychain variable framework of the antibody produced by the hybridoma cellline having an ATCC Accession Number HB-12126 or 12109. In otherembodiments, the non-human heavy chain variable framework is from murineJ591 antibody (SEQ ID NO:7; see FIG. 1A), from murine J415 antibody (SEQID NO:35; see FIG. 5), from murine J533 antibody (SEQ ID NO:109; seeFIG. 9A), or from murine E99 antibody (SEQ ID NO:119; see FIG. 11A), orthe heavy chain variable framework of the antibody produced by thehybridoma cell line having an ATCC Accession Number HB-12126, HB-12109,HB-12127 or HB-12101.

In yet other embodiments, the heavy chain variable framework of themodified anti-PSMA antibody, or antigen-binding fragment thereof,includes a non-human (e.g., a murine) heavy chain variable framework(e.g., a murine J591 heavy chain variable framework (SEQ ID NO:7, asshown FIG. 1A, or the heavy chain variable framework of the antibodyproduced by the hybridoma cell line having an ATCC Accession NumberHB-12126) which has at least 3, 5, 10, 15, 16, 17, 18, or 19 amino acidreplacements. In one embodiment, the non-human heavy chain variableframework of the modified anti-PSMA antibody includes one or more of:

-   -   a framework region 1 having at least 4, 5, or 6 replacements;    -   a framework region 2 having at least 1, 2, or 3 replacements;    -   a framework region 3 having at least 3, 4, or 5 replacements; or    -   a framework region 4 having at least one replacement.

In yet other embodiments, the heavy chain variable framework of themodified anti-PSMA antibody, or antigen-binding fragment thereof,includes a non-human (e.g., a murine) heavy chain variable framework(e.g., a murine J415 heavy chain variable framework (SEQ ID NO:35, asshown in FIG. 5, or the heavy chain variable framework of the antibodyproduced by the hybridoma cell line having an ATCC Accession NumberHB-12109) which has at least 1, 2, 3, 4, or 5 amino acid replacements.In one embodiment, the non-human heavy chain variable framework of themodified anti-PSMA antibody includes one or more of:

-   -   a framework region 1 having at least one replacement;    -   a framework region 3 having at least 1, 2, or 3 replacements; or    -   a framework region 4 having at least one replacement.

The replacement can be chosen from: a conservative substitution of anon-human residue, or a residue found in a human germline, mature orconsensus sequence at the same position, e.g. the most common residue inthe human germline at the same position. In one embodiment, the heavychain variable framework has at least 3, 4, 5, 6 and preferably 7conservative substitutions. Preferably, the heavy chain variableframework has at least 5, 6, 7 and preferably 8 replacements by the mostcommon residue in the human germline at the same position.

In some embodiments, the non-human heavy chain variable framework has atleast one amino acid replacement at a position selected from the groupconsisting of: residue 5, 11, 12, 16, 17, 19, 40, 41, 44, 75, 76, 82a,83, 87, and 108 (Kabat numbering as shown in Table 3). The replacementcan be chosen from one or more of: 5 (valine), 11 (valine), 12 (lysine),16 (alanine), 17 (threonine), 19 (lysine), 40 (alanine), 41 (proline),44 (glycine), 75 (threonine), 76 (aspartate), 82a (serine), 83(arginine), 87 (threonine), and 108 (leucine) (Kabat numbering as shownin Table 4).

In other embodiments, the non-human heavy chain variable framework hasat least one amino acid replacement at a position selected from thegroup consisting of: residue 20, 87, 94, 95 and 112 (linear numbering asshown in Table 5). The replacement can be chosen from one or more of:residue 20 (isoleucine), 87 (serine), 94 (alanine), 95 (valine), and 112(valine) (linear numbering as shown in Table 5).

The amino acid sequence of the framework regions of the light and heavychains regions of antibodies J591, J415, J533 and E99 are provided inTable 6, below.

TABLE 6 Framework Sequences SEQ ID NAME Organism FIG. NO: SEQUENCEV_(H) FR1-FR4 Mus musculus FIG. 1A   7 EVQLQQSGPELKKPGTSVRISCK J591TSWVKQSHGKSLEWIGKATLTV DKSSSTAYMELRSLTSEDSAVY YCAAWGQGTTLTVSSV_(L) FR1-FR4 Mus musculus FIG. 1B   8 DIVMTQSHKFMSTSVGDRVSIIC J591WYQQKPGQSPKLLIYGVPDRFT GSGSGTDFTLTITNVQSEDLADY FCFGAGTMLDLKV_(H) FR1 (Deimm) Artificial- FIG. 2A   9 EVQLVQSGPEVKKPGATVKISC J591deimmunized KTS heavy chain J591 V_(H) FR2 (Deimm) Artificial- FIG. 2A 10 WVKQAPGKGLEWIG J591 deimmunized heavy chain J591 V_(H) FR3 (Deimm)Artificial- FIG. 2A  11 KATLTVDKSTDTAYMELSSLRS J591 deimmunizedEDTAVYYCAA heavy chain J591 V_(H) FR4 (Deimm) Artificial- FIG. 2A  12WGQGTLLTVSS J591 deimmunized heavy chain J591 V_(L) FR1 (Deimm)Artificial- FIG. 2B  13 DIQMTQSPSSLSTSVGDRVTLTC J591 deimmunizedlight chain J591 V_(L) FR2 (Deimm) Artificial- FIG. 2B  14WYQQKPGPSPKLLIY J591 deimmunized light chain J591 V_(L) FR3 (Deimm)Artificial- FIG. 2B  15 GIPSRFSGSGSGTDFTLTISSLQPE J591 deimmunizedDFADYYC light chain J591 V_(L) FR4 (Deimm) Artificial- FIG. 2B  16FGPGTKVDIK J591 deimmunized light chain J591 V_(H) FR1-FR4 Artificial-FIG. 2A  17 EVQLVQSGPEVKKPGATVKISC (Deimm) deimmunizedKTSWVKQAPGKGLEWIGKATLT J591 heavy chain VDKSTDTAYMELSSLRSEDTAV J591YYCAAWGQGTLLTVSS V_(L)FR1-FR4 Artificial- FIG. 2B  18DIQMTQSPSSLSTSVGDRVTLTC (Deimm) deimmunized WYQQKPGPSPKLLIYGIPSRFSGSJ591 light chain GSGTDFTLTISSLQPEDFADYYC J591 FGPGTKVDIK V_(H)FR1-FR4Mus musculus FIG. 5  35 EVKLEESGGGLVQPGGSMKLSC J415VASWVRQSPEKGLEWVARVIIS RDDSKSSVYLQMNNLRAEDTGI YYCTRWGQGTTLTVSSV_(L) FR1-FR4 Mus musculus FIG. 6  36 NIVMTQFPKSMSISVGERVTLTC J415WYQQKPEQSPKMLIYGVPDRFT GSGSATDFILTISSVQTEDLVDY YCFGGGTKLEMKV_(H) FR1 (Deimm) Artificial- FIG. 5  37 EVKLEESGGGLVQPGGSMKISC J415-4deimmunized VAS heavy chain J415-4 V_(H) FR2 (Deimm) Artificial- FIG. 5 38 WVRQSPEKGLEWVA J415-4 deimmunized heavy chain J415-4V_(H) FR3 (Deimm) Artificial- FIG. 5  39 RVIISRDDSKSSVYLQMNSLRAE J415-4deimmunized DTAVYYCTR heavy chain J415-4 V_(H) FR4 (Deimm) Artificial-FIG. 5  40 WGQGTTVTVSS J415-4 deimmunized heavy chain J415-4V_(L) FR1 (Deimm) Artificial- FIG. 6  41 NIVMTQFPKSMSASAGERMTLT J415-5deimmunized C light chain J415-5 V_(L) FR2 (Deimm) Artificial- FIG. 6 42 WYQQKPTQSPKMLIY J415-5 deimmunized light chain J415-5V_(L) FR3 (Deimm) Artificial- FIG. 6  43 GVPDRFSGSGSGTDFILTISSVQA J415-5deimmunized EDLVDYYC light chain J415-5 V_(L) FR4 (Deimm) Artificial-FIG. 6  44 FGGGTKLEMK J415-5 deimmunized light chain J415-5V_(H) FR1-FR4 Artificial- FIG. 5  45 EVKLEESGGGLVQPGGSMKISC (Deimm)deimmunized VASWVRQSPEKGLEWVARVIIS J415-4 heavy chainRDDSKSSVYLQMNSLRAEDTAV J415-4 YYCTRWGQGTTVTVSS V_(L) FR1-FR4 Artificial-FIG. 6  46 NIVMTQFPKSMSASAGERMTLT (Deimm) deimmunizedCWYQQKPTQSPKMLIYGVPDRF J415-5 light chain SGSGSGTDFILTISSVQAEDLVDYJ415-5 YCFGGGTKLEMK V_(H) FR1 Mus musculus FIG. 9A 105EVQLQQSGPELVKPGASVKMSC J533 KAS V_(H) FR2 Mus musculus FIG. 9A 106WVKQKPGQVLEWIG J533 V_(H) FR3 Mus musculus FIG. 9A 107KATLTSDKYSSTAYMELSGLTSE J533 DSAVYYCAR V_(H) FR4 Mus musculus FIG. 9A108 WGRGATLTVSS J533 V_(H) FR1-FR4 Mus musculus FIG. 9A 109EVQLQQSGPELVKPGASVKMSC J533 KASWVKQKPGQVLEWIGKATLTSDKYSSTAYMELSGLTSEDSAV YYCARWGRGATLTVSS V_(L) FR1 Mus musculus FIG. 10A110 DIVLTQSPASLAVSLGQRATISC J533 V_(L) FR2 Mus musculus FIG. 10A 111WYQQKPGQPPNLLIF J533 V_(L) FR3 Mus musculus FIG. 10A 112GIPARFSGSGSGTDFTLTIYPVEA J533 DDVATYYC V_(L) FR4 Mus musculus FIG. 10A113 FGGGTKLEIK J533 V_(L) FR1-FR4 Mus musculus FIG. 10A 114DIVLTQSPASLAVSLGQRATISC J533 WYQQKPGQPPNLLIFGIPARFSGSGSGTDFTLTIYPVEADDVATYY CFGGGTKLEIK V_(H) FR1 Mus musculus FIG. 11A 115QVQLKESGPGLVASSQSLSITCT E99 VS V_(H) FR2 Mus musculus FIG. 11A 116WVRQPPGKGLEWLG E99 V_(H) FR3 Mus musculus FIG. 11A 117RLNIFKDNSKNQVFLKMSSFQTD E99 DTARYFCAR V_(H) FR4 Mus musculus FIG. 11A118 WGQGTTLTVSS E99 V_(H) FR1-FR4 Mus musculus FIG. 11A 119QVQLKESGPGLVASSQSLSITCT E99 VSWVRQPPGKGLEWLGRLNIFKDNSKNQVFLKMSSFQTDDTARY FCARWGQGTTLTVSS V_(L) FR1 Mus musculus FIG. 12A120 NIVMTQSQKFMSTSPGDRVRVT E99 C V_(L) FR2 Mus musculus FIG. 12A 121WYQAKPGQSPRILIY E99 V_(L) FR3 Mus musculus FIG. 12A 122GVPDRFTAYGSGTDFTLTITNVQ E99 SEDLTEYFC V_(L) FR4 Mus musculus FIG. 12A123 FGAGTKLELK E99 V_(L) FR1-FR4 Mus musculus FIG. 12A 124NIVMTQSQKFMSTSPGDRVRVT E99 CWYQAKPGQSPRILIYGVPDRFTAYGSGTDFTLTITNVQSEDLTEY FCFGAGTKLELK

In other embodiments, the anti-PSMA antibody, or antigen-bindingfragment thereof, includes at least one light chain or heavy chainimmuoglobulin or, preferably, at least one light chain immunoglobulinand at least one heavy chain immunoglobulin. Preferably, the light chainimmunoglobulin includes a non-human light chain variable regioncomprising three CDRs from a non-human, e.g., murine, anti-PSMA lightchain variable region (e.g., the murine J591 or J415 light chainvariable region shown in SEQ ID NO:20 (see FIG. 1B) or SEQ ID NO:48 (seeFIG. 6), respectively, or the light chain variable region of theantibody produced by the hybridoma cell line having an ATCC AccessionNumber HB-12126 or 12109) and a light chain framework which differs fromthe framework of the non-human, e.g., murine, anti-PSMA light chainframework (e.g., the murine J591 of J415 light chain framework shown inSEQ ID NO:8 (see FIG. 1B) or SEQ ID NO:36 (see FIG. 6), respectively, orthe light chain variable framework of the antibody produced by thehybridoma cell line having an ATCC Accession Number HB-12126 or 12109)at one, two, three, four, five, six, seven or more positions selectedfrom the group consisting of: residue 3, 8, 9, 10, 11, 20, 21, 22, 42,58, 60, 63, 76, 77, 78, 80, 83, 87, 100, 103, 104 and 106 (Kabatnumbering as in Table 2), or residues 13, 15, 19, 41, 63, 68, and 80(linear numbering as in Table 3).

In other preferred embodiments, the heavy chain immunoglobulin includesa non-human heavy chain variable region comprising three complementaritydetermining regions (CDRs) from a non-human, e.g., murine, anti-PSMAheavy chain variable region (e.g., the murine J591 or J415 heavy chainvariable region shown in SEQ ID NO:19 (see FIG. 1A) or SEQ ID NO:47 (seeFIG. 5), respectively, or the heavy chain variable region of theantibody produced by the hybridoma cell line having an ATCC AccessionNumber HB-12126 or HB-12109) and a modified heavy chain framework whichdiffers from the framework of the non-human, e.g., murine, anti-PSMAheavy chain framework (e.g., the murine J591 or J415 heavy chainframework shown in SEQ ID NO:7 (see FIG. 1A) or SEQ ID NO:35 (see FIG.5), respectively, or the heavy chain variable framework of the antibodyproduced by the hybridoma cell line having an ATCC Accession NumberHB-12126 or HB-12109) at one, two, three, four, five or more positionsselected from the group consisting of: residue 5, 11, 12, 16, 17, 19,40, 41, 44, 75, 76, 82a, 83, 87, and 108 (Kabat numbering as in Table4), or residue 20, 87, 94, 95 and 112 (linear numbering as in Table 5).

In yet other embodiments, the modified anti-PSMA antibody, orantigen-binding fragment thereof, includes at least one light or heavychain immunoglobulin or, more preferably, at least one light chainimmunoglobulin and at least one heavy chain immunoglobulin. Preferably,the light chain immunoglobulin includes a modified non-human light chainvariable region comprising three CDRs from a non-human, e.g., murine,anti-PSMA light chain variable region (e.g., the murine J591 light chainvariable region shown in SEQ ID NO:20 (see FIG. 1B), or the light chainvariable region of the antibody produced by the hybridoma cell linehaving an ATCC Accession Number HB-12126) and a modified light chainframework which differs from the framework of the non-human anti-PSMAlight chain variable region, e.g., the murine J591 light chain variableregion (SEQ ID NO:20 or the light chain variable region of the antibodyproduced by the hybridoma cell line having an ATCC Accession NumberHB-12126), by at least one, two, three, four, five, six, seven, eight,nine, ten positions selected from the group consisting of:

-   -   a position within or adjacent to one or more of residues 1, 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, or a T cell epitope which        includes one or more of residues 1-13 (numbering as in FIG. 3B);    -   a position within or adjacent to one or more of residues 8, 9,        10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or a T cell        epitope which includes one or more of residues 8-20 (numbering        as in FIG. 3B);    -   a position within or adjacent to one or more of residues 17, 18,        19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29, or a T cell        epitope which includes one or more of residues 17-29 (numbering        as in FIG. 3B);    -   a position within or adjacent to one or more of residues 27, 28,        29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39, or a T cell        epitope which includes one or more of residues 27-39 (numbering        as in FIG. 3B);    -   a position within or adjacent to one or more of residues 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43, or a T cell        epitope which includes one or more of residues 30-43 (numbering        as in FIG. 3B);    -   a position within or adjacent to one or more of residues 45, 46,        47, 48, 49, 50, 51, 52, 53, 54, 55, 56, or 57, or a T cell        epitope which includes one or more of residues 45-57 (numbering        as in FIG. 3B);    -   a position within or adjacent to one or more of residues 56, 57,        58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68, or a T cell        epitope which includes one or more of residues 56-68 (numbering        as in FIG. 3B);    -   a position within or adjacent to one or more of residues 71, 72,        73, 74, 75, 76, 77, 78, 79, 80, 81, 82, or 83, or a T cell        epitope which includes one or more of residues 71-83 (numbering        as in FIG. 3B);    -   a position within or adjacent to one or more of residues 73, 74,        75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or 85, or a T cell        epitope which includes one or more of residues 73-85 (numbering        as in FIG. 3B); and    -   a position within or adjacent to one or more of residues 94, 95,        96, 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106, or a T        cell epitope which includes one or more of residues 94-106        (numbering as in FIG. 3B).

In yet other embodiments, the anti-PSMA antibody, or antigen-bindingfragment thereof, includes at least one light or heavy chainimmunoglobulin or, more preferably, at least one light chainimmunoglobulin and at least one modified heavy chain immunoglobulin.Preferably, the light chain immunoglobulin includes a modified non-humanlight chain variable region comprising three CDRs from a non-human,e.g., murine, anti-PSMA light chain variable region (e.g., the murineJ415 light chain variable region shown in SEQ ID NO:48 (FIG. 37), or thelight chain variable region of the antibody produced by the hybridomacell line having an ATCC Accession Number HB-12109) and a light chainframework which differs from the framework of the non-human anti-PSMAlight chain variable region, e.g., the murine J415 light chain variableregion (SEQ ID NO:48 or the light chain variable region of the antibodyproduced by the hybridoma cell line having an ATCC Accession NumberHB-12109), by at least one, two, three, four, five, six, seven positionsselected from the group consisting of:

-   -   a position within or adjacent to one or more of residues 5, 6,        7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18, or a T cell        epitope which includes one or more of residues 5-18 (linear        numbering as in FIG. 6);    -   a position within or adjacent to one or more of residues 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24, or a T cell        epitope which includes one or more of residues 11-24 (linear        numbering as in FIG. 6);    -   a position within or adjacent to one or more of residues 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, or a T cell        epitope which includes one or more of residues 13-26 (linear        numbering as in FIG. 6);    -   a position within or adjacent to one or more of residues 17, 18,        19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or a T cell        epitope which includes one or more of residues 17-30 (linear        numbering as in FIG. 6);    -   a position within or adjacent to one or more of residues 27, 28,        29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or a T cell        epitope which includes one or more of residues 27-40 (linear        numbering as in FIG. 6);    -   a position within or adjacent to one or more of residues 31, 32,        33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44, or a T cell        epitope which includes one or more of residues 31-44 (linear        numbering as in FIG. 6);    -   a position within or adjacent to one or more of residues 56, 57,        58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69, or a T cell        epitope which includes one or more of residues 56-69 (linear        numbering as in FIG. 6);    -   a position within or adjacent to one or more of residues 60, 61,        62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73, or a T cell        epitope which includes one or more of residues 60-73 (linear        numbering as in FIG. 6);    -   a position within or adjacent to one or more of residues 70, 71,        72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, or 83, or a T cell        epitope which includes one or more of residues 70-83 (linear        numbering as in FIG. 6);    -   a position within or adjacent to one or more of residues 71, 72,        73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 or 84, or a T cell        epitope which includes one or more of residues 71-84 (linear        numbering as in FIG. 6);    -   a position within or adjacent to one or more of residues 73, 74,        75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 or 86, or a T cell        epitope which includes one or more of residues 73-86 (linear        numbering as in FIG. 6);    -   a position within or adjacent to one or more of residues 76, 77,        78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, or 92,        or a T cell epitope which includes one or more of residues 76-92        (linear numbering as in FIG. 6); and    -   a position within or adjacent to one or more of residues 81, 82,        83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 94, or a T cell        epitope which includes one or more of residues 81-94 (linear        numbering as in FIG. 6).    -   In other embodiments, the heavy chain immunoglobulin of the        anti-PSMA antibody, or antigen-binding fragment thereof,        includes a non-human heavy chain variable region comprising        three CDRs from a non-human, e.g., murine, anti-PSMA heavy chain        variable region (e.g., the murine J591 heavy chain variable        region shown in SEQ ID NO:19 (see FIG. 1 A), or the heavy chain        variable region of the antibody produced by the hybridoma cell        line having an ATCC Accession Number HB-12126) and a heavy chain        framework which differs from the framework of the non-human        anti-PSMA heavy chain variable region (e.g., the murine J591        heavy chain variable region of SEQ ID NO:19 or the heavy chain        variable framework of the antibody produced by the hybridoma        cell line having an ATCC Accession Number HB-12126), by at least        one, two, three, five, seven, ten positions selected from the        group consisting of:    -   a position within or adjacent to one or more of residues 2, 3,        4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, or a T cell epitope        which includes one or more of residues 2-14 (numbering as in        FIG. 3A);    -   a position within or adjacent to one or more of residues 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, or a T cell        epitope which includes one or more of residues 10-22 (numbering        as in FIG. 3A);    -   a position within or adjacent to one or more of residues 16, 17,        18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28, or a T cell        epitope which includes one or more of residues 16-28 (numbering        as in FIG. 3A);    -   a position within or adjacent to one or more of residues 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42, or a T cell        epitope which includes one or more of residues 30-42 (numbering        as in FIG. 3A);    -   a position within or adjacent to one or more of residues 32, 33,        34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44, or a T cell        epitope which includes one or more of residues 32-44 (numbering        as in FIG. 3A);    -   a position within or adjacent to one or more of residues 43, 44,        45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55, or a T cell        epitope which includes one or more of residues 43-55 (numbering        as in FIG. 3A);    -   a position within or adjacent to one or more of residues 46, 47,        48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58, or a T cell        epitope which includes one or more of residues 46-58 (numbering        as in FIG. 3A);    -   a position within or adjacent to one or more of residues 58, 59,        60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70, or a T cell        epitope which includes one or more of residues 58-70 (numbering        as in FIG. 3A);    -   a position within or adjacent to one or more of residues 62, 63,        64, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 74, or a T cell        epitope which includes one or more of residues 62-74 (numbering        as in FIG. 3A);    -   a position within or adjacent to one or more of residues 70, 71,        72, 73, 74, 75, 76, 77, 78, 79, 80 or 81, or a T cell epitope        which includes one or more of residues 70-81 (numbering as in        FIG. 3A);    -   a position within or adjacent to one or more of residues 81, 82,        83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93, or a T cell        epitope which includes one or more of residues 81-93 (numbering        as in FIG. 3A);    -   a position within or adjacent to one or more of residues 84, 85,        86, 87, 88, 89, 90, 91, 92, 93, 95, or 96, or a T cell epitope        which includes one or more of residues 84-96 (numbering as in        FIG. 3A);    -   a position within or adjacent to one or more of residues 91, 92,        93, 95, 96, 97, 98, 99, 100, 101, 102, or 103, or a T cell        epitope which includes one or more of residues 91-103 (numbering        as in FIG. 3A); and    -   a position within or adjacent to one or more of residues 100,        101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 112,        or a T cell epitope which includes one or more of residues        100-112 (numbering as in FIG. 3A).

In other embodiments, the heavy chain immunoglobulin of the anti-PSMAantibody, or antigen-binding fragment thereof, includes a non-humanheavy chain variable region comprising three CDRs from a non-human,e.g., murine, anti-PSMA heavy chain variable region (e.g., the murineJ415 heavy chain variable region shown in SEQ ID NO:47 (see FIG. 5), orthe heavy chain variable region of the antibody produced by thehybridoma cell line having an ATCC Accession Number HB-12109) and aheavy chain framework which differs from the framework of the non-humananti-PSMA heavy chain variable region, e.g., the murine J591 heavy chainvariable region of SEQ ID NO:47 or the heavy chain variable framework ofthe antibody produced by the hybridoma cell line having an ATCCAccession Number HB-12109), by at least one, two, three, four, fivepositions selected from the group consisting of:

-   -   a position within or adjacent to one or more of residues 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, or a T cell        epitope which includes one or more of residues 10-23 (numbering        as in FIG. 5);    -   a position within or adjacent to one or more of residues 16, 17,        18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29, or a T cell        epitope which includes one or more of residues 16-29 (numbering        as in FIG. 5);    -   a position within or adjacent to one or more of residues 21, 22,        23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34, or a T cell        epitope which includes one or more of residues 21-34 (numbering        as in FIG. 5);    -   a position within or adjacent to one or more of residues 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43, or a T cell        epitope which includes one or more of residues 30-43 (numbering        as in FIG. 5);    -   a position within or adjacent to one or more of residues 35, 36,        37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or a T cell        epitope which includes one or more of residues 35-48 (numbering        as in FIG. 5);    -   a position within or adjacent to one or more of residues 43, 44,        45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or 56, or a T cell        epitope which includes one or more of residues 43-56 (numbering        as in FIG. 5);    -   a position within or adjacent to one or more of residues 46, 47,        48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 or 59, or a T cell        epitope which includes one or more of residues 46-59 (numbering        as in FIG. 5);    -   a position within or adjacent to one or more of residues 49, 50,        51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62, or a T cell        epitope which includes one or more of residues 49-62 (numbering        as in FIG. 5);    -   a position within or adjacent to one or more of residues 64, 65,        66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, or 77, or a T cell        epitope which includes one or more of residues 64-77 (numbering        as in FIG. 5);    -   a position within or adjacent to one or more of residues 80, 81,        82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93, or a T cell        epitope which includes one or more of residues 80-93 (numbering        as in FIG. 5);    -   a position within or adjacent to one or more of residues 86, 87,        88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, or a T cell        epitope which includes one or more of residues 86-99 (numbering        as in FIG. 5); and    -   a position within or adjacent to one or more of residues 104,        105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, or        117, or a T cell epitope which includes one or more of residues        104-117 (numbering as in FIG. 5).

In yet other embodiments, the anti-PSMA antibody, or antigen-bindingfragment thereof, includes at least one light or heavy chainimmunoglobulin or, more preferably, at least one light chainimmunoglobulin and at least one heavy chain immunoglobulin. Preferably,the light chain immunoglobulin includes a non-human light chain variableregion comprising three CDRs from a non-human, e.g., murine, anti-PSMAlight chain variable region (e.g., the murine J591 light chain variableregion shown in SEQ ID NO:20 (FIG. 1B), or the light chain variableregion of the antibody produced by the hybridoma cell line having anATCC Accession Number HB-12126) and a light chain framework whichdiffers from the framework of the non-human anti-PSMA light chainvariable region, e.g., murine J591 light chain variable region, by atleast one position while having a residue from the non-human anti-PSMAlight chain variable region at at least one, two, three, five, seven,ten, fifteen, or twenty residues selected from the group consisting of1, 2, 4-7, 12-19, 23, 31-41, 43-49, 57, 59, 61, 62, 64-75, 79, 82, 83,85-87, 89, 98, 99, 101, 102, 105, and 106 (numbering as in FIG. 3B). Thelight chain framework can differ at positions chosen from one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, nineteen, twenty or more residuesselected from the group consisting of 3, 8, 9, 10, 11, 20, 21, 22, 42,58, 60, 63, 76, 77, 78, 80, 83, 87, 100, 103, and 104 (numbering as inFIG. 3B).

In yet other embodiments, the anti-PSMA antibody, or antigen-bindingfragment thereof, includes at least one light or heavy chainimmunoglobulin or, more preferably, at least one light chainimmunoglobulin and at least one heavy chain immunoglobulin. Preferably,the modified light chain immunoglobulin includes a non-human light chainvariable region comprising three CDRs from a non-human, e.g., murine,anti-PSMA light chain variable region (e.g., the murine J415 light chainvariable region shown in SEQ ID NO:48 (FIG. 6), or the light chainvariable region of the antibody produced by the hybridoma cell linehaving an ATCC Accession Number HB-12109) and a light chain frameworkwhich differs from the framework of the non-human anti-PSMA light chainvariable region, e.g., murine J415 light chain variable region, by atleast one position while having a residue from the non-human anti-PSMAlight chain variable region at at least one, two, three, five, seven,ten, fifteen, or twenty residues selected from the group consisting of1-12, 14, 16-18, 20-40, 42-62, 64-67, 69-79, and 81-107 (linearnumbering as in FIG. 6). The modified light chain framework can differat at least one, two, three, four, five, six, or seven positionsselected from the group consisting of 13, 15, 19, 41, 63, 68 and 80(linear numbering as in FIG. 6).

In other embodiments, the heavy chain immunoglobulin of the modifiedanti-PSMA antibody, or antigen-binding fragment thereof, includes anon-human heavy chain variable region comprising three CDRs from anon-human, e.g., murine, anti-PSMA heavy chain variable region (e.g.,the murine J591 heavy chain variable region shown in SEQ ID NO:19 (FIG.1A), or the heavy chain variable region of the antibody produced by thehybridoma cell line having an ATCC Accession Number HB-12126) and amodified heavy chain framework which differs from the framework of thenon-human anti-PSMA heavy chain variable region by at least one positionwhile having a residue from the non-human anti-PSMA heavy chain variableregion at at least one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, or fourteen residues selected from thegroup consisting of 1-4, 6-10, 13-15, 18, 20-25, 36-39, 42, 43, 45-49,67-75, 78-83, 85, 86, 88-90, 92-98, 105-109, and 111-115 (numbering asin FIG. 3A). The modified heavy chain framework can differ at at leastone, two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, or fourteen positions selected from the groupconsisting of 5, 11-12, 16-17, 19, 26-35, 40-41, 44, 50-66, 76-77, 84,87, 91, 99-104, and 110 (numbering as in FIG. 3A).

In other embodiments, the heavy chain immunoglobulin of the anti-PSMAantibody, or antigen-binding fragment thereof, includes a non-humanheavy chain variable region comprising three CDRs from a non-human,e.g., murine, anti-PSMA heavy chain variable region (e.g., the murineJ415 heavy chain variable region shown in SEQ ID NO:47 (FIG. 5), or theheavy chain variable region of the antibody produced by the hybridomacell line having an ATCC Accession Number HB-12109) and a heavy chainframework which differs from the framework of the non-human anti-PSMAheavy chain variable region by at least one position while having aresidue from the non-human anti-PSMA heavy chain variable region at atleast one, two, three, four, or five residues selected from the groupconsisting of 1-19, 21-86, 88-93, 96-111, and 113-116 (numbering as inFIG. 5). The heavy chain framework can differ at a positions selectedfrom the group consisting of 20, 87, 94, 95 and 112 (numbering as inFIG. 5).

In yet another aspect, the heavy chain immunoglobulin of the anti-PSMAantibody, or antigen-binding fragment thereof, includes a heavy chainvariable region comprising at least one, two, three, four, five, six,seven, eight, nine, ten, twenty, twenty-five, thirty, thirty-five,forty, forty-five, or fifty amino acid residues chosen from one or moreof the following residues and located at a position chosen from one ormore of: residue 1 (glutamate), 2 (valine), 4 (leucine), 7 (serine), 8(glycine), 11 (leucine), 14 (proline), 15 (glycine), 19 (lysine), 20(isoleucine), 21 (serine), 22 (cysteine), 25 (serine), 26 (glycine), 28(threonine), 29 (phenylalanine), 32 (tyrosine), 36 (tryptophan), 37(valine), 38 (arginine/lysine), 39 (glutamine), 41 (proline), 43(lysine), 44 (glycine), 45 (leucine), 46 (glutamate), 47 (tryptophan),51 (isoleucine), 67 (arginine/lysine), 73 (aspartate), 75 (serine), 80(tyrosine), 85 (serine), 86 (leucine), 87 (arginine), 89 (glutamate), 90(aspartate), 91 (threonine), 92 (alanine), 93 (valine), 94 (tyrosine),95 (tyrosine), 96 (cysteine), 100 (tryptophan), 101 (asparagine), 105(tryptophan), 106 (glycine), 107 (glutamine), 108 (glycine), 109(threonine), 112 (threonine), 113 (valine), 114 (serine), or 115(serine) (linear numbering as shown in FIG. 3A).

In one embodiment, the heavy chain immunoglobulin of the anti-PSMAantibody, or antigen-binding fragment thereof, includes one or more of:

-   -   a framework region 1 having at least one, two, three, four,        five, six, seven, eight, nine, ten, eleven, twelve, thirteen        amino acids selected from the group consisting of residue 1        (glutamate), 2 (valine), 4 (leucine), 7 (serine), 8 (glycine),        11 (leucine), 14 (proline), 15 (glycine), 19 (lysine), 20        (isoleucine), 21 (serine), 22 (cysteine), and 25 (serine)        (linear numbering as shown in FIG. 3A);    -   a CDR1 having at least one, two, three, four amino acids        selected from the group consisting of residue 26 (glycine), 28        (threonine), 29 (phenylalanine), and 32 (tyrosine) (linear        numbering as shown in FIG. 3A);    -   a framework region 2 having at least one, two, three, four,        five, six, seven, eight, nine, ten amino acids selected from the        group consisting of residue 36 (tryptophan), 37 (valine), 38        (arginine/lysine), 39 (glutamine), 41 (proline), 43 (lysine), 44        (glycine), 45 (leucine), 46 (glutamate), and 47 (tryptophan)        (linear numbering as shown in FIG. 3A);    -   a CDR2 having at least one isoleucine at position 51 (linear        numbering as shown in FIG. 3A);    -   a framework region 3 having at least one, two, three, four,        five, six, seven, eight, nine, ten, eleven, twelve, thirteen,        fourteen amino acids selected from the group consisting of        residue 67 (arginine/lysine), 73 (aspartate), 75 (serine), 80        (tyrosine), 85 (serine), 86 (leucine), 87 (arginine), 89        (glutamate), 90 (aspartate), 91 (threonine), 92 (alanine), 93        (valine), 94 (tyrosine), 95 (tyrosine), and 96 (cysteine)        (linear numbering as shown in FIG. 3A);    -   a CDR3 having at least one, two amino acids selected from the        group consisting of residue 100 (tryptophan) and 101        (asparagine) (linear numbering as shown in FIG. 3A); or    -   a framework region 4 having at least one, two, three, four,        five, six, seven, eight, nine amino acids selected from the        group consisting of residue 105 (tryptophan), 106 (glycine), 107        (glutamine), 108 (glycine), 109 (threonine), 112 (threonine),        113 (valine), 114 (serine), and 115 (serine) (linear numbering        as shown in FIG. 3A).

In yet another embodiment, the light chain immunoglobulin of themodified anti-PSMA antibody, or antigen-binding fragment thereof,includes a light chain variable region comprising at least one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, twenty, thirty, forty, fifty, sixty, or seventyamino acids chosen from one or more of the following residues andlocated at a position chosen from one or more of: residue 2(isoleucine), 4 (methionine), 5 (threonine), 6 (glutamine), 8 (proline),10 (serine), 12 (serine), 14 (serine), 16 (glycine), 17(glutamate/aspartate), 18 (arginine), 20 (threonine), 21 (leucine), 22(threonine), 23 (cysteine), 24 (lysine), 25 (alanine), 26 (serine), 29(valine), 30 (glycine), 31 (threonine), 33 (valine), 35 (tryptophan), 36(tyrosine), 37 (glutamine), 38 (glutamine), 39 (lysine), 40 (proline),43 (serine), 44 (proline), 45 (lysine), 47 (leucine), 48 (isoleucine),49 (tyrosine), 51 (alanine), 52 (serine), 54 (arginine), 56 (threonine),57 (glycine), 59 (proline), 61 (arginine), 62 (phenylalanine), 63(serine), 64 (glycine), 65 (serine), 66 (glycine), 67 (serine), 68(glycine), 69 (threonine), 70 (aspartate), 71 (phenylalanine), 73(leucine), 74 (threonine), 75 (threonine), 76 (serine), 77 (serine), 79(glutamine), 81 (glutamate), 82 (aspartate), 85 (aspartate), 86(tyrosine), 87 (tyrosine), 88 (cysteine), 90 (glutamine), 95 (proline),97 (threonine), 98 (phenylalanine), 99 (glycine), 101 (glycine), 102(threonine), 103 (lysine), 105 (glutamate/aspartate), or 107 (lysine)(linear numbering as in FIG. 3B).

In one embodiment, the light chain immunoglobulin of the anti-PSMAantibody, or antigen-binding fragment thereof, includes one or more of:

-   -   a framework region 1 having at least one, two, three, four,        five, six, seven, eight, nine, ten, eleven, twelve, fourteen,        fifteen amino acids selected from the group consisting of        residue 2 (isoleucine), 4 (methionine), 5 (threonine), 6        (glutamine), 8 (proline), 10 (serine), 12 (serine), 14 (serine),        16 (glycine), 17 (glutamate/aspartate), 18 (arginine), 20        (threonine), 21 (leucine), 22 (threonine), and 23 (cysteine)        (linear numbering as shown in FIG. 3B);    -   a CDR1 having at least one, two, three, four, five, six, seven        amino acids selected from the group consisting of residue 24        (lysine), 25 (alanine), 26 (serine), 29 (valine), 30 (glycine),        31 (threonine), and 33 (valine) (linear numbering as shown in        FIG. 3B);    -   a framework region 2 having at least one, two, three, four,        five, six, seven, eight, nine, ten, eleven, twelve amino acids        selected from the group consisting of residue 35 (tryptophan),        36 (tyrosine), 37 (glutamine), 38 (glutamine), 39 (lysine), 40        (proline), 43 (serine), 44 (proline), 45 (lysine), 47 (leucine),        48 (isoleucine), and 49 (tyrosine) (linear numbering as shown in        FIG. 3B);    -   a CDR2 having at least one, two, three, four amino acids        selected from the group consisting of residue 51 (alanine), 52        (serine), 54 (arginine), and 56 (threonine) (linear numbering as        shown in FIG. 3B);    -   a framework region 3 having at least one, two, three, four,        five, six, seven, eight, nine, ten, eleven, twelve, thirteen,        fourteen, fifteen, twenty, twenty-one, twenty-two, twenty-three,        twenty-four amino acids selected from the group consisting of        residue 59 (proline), 61 (arginine), 62 (phenylalanine), 63        (serine), 64 (glycine), 65 (serine), 66 (glycine), 67 (serine),        68 (glycine), 69 (threonine), 70 (aspartate), 71        (phenylalanine), 73 (leucine), 74 (threonine), 75 (threonine),        76 (serine), 77 (serine), 79 (glutamine), 81 (glutamate), 82        (aspartate), 85 (aspartate), 86 (tyrosine), 87 (tyrosine), and        88 (cysteine) (linear numbering as shown in FIG. 3B);    -   a CDR3 having at least one, two, three, four amino acids        selected from the group consisting of residue 90 (glutamine), 95        (proline), 97 (threonine), and 98 (phenylalanine) (linear        numbering as shown in FIG. 3B); or    -   a framework region 4 having at least one, two, three, four,        five, six amino acid selected from the group consisting of        residue 99 (glycine), 101 (glycine), 102 (threonine), 103        (lysine), 105 (glutamate/aspartate), and 107 (lysine) (linear        numbering as shown in FIG. 3B).

An anti-PSMA antibody, e.g., a modified anti-PSMA antibody, orantigen-binding fragment thereof, described herein can be used alone,e.g., can be administered to a subject, or used in vitro, innon-derivatized or unconjugated forms. In other embodiments, theanti-PSMA antibody, or antigen-binding fragment thereof, can bederivatized or linked to another molecular entity, typically a label ora therapeutic (e.g., a cytotoxic or cytostatic) agent. The molecularentity can be, e.g., another peptide, protein (including, e.g., a viralcoat protein of, e.g., a recombinant viral particle), a non-peptidechemical compound, isotope, etc. The anti-PSMA antibody, orantigen-binding fragment thereof, can be functionally linked, e.g., bychemical coupling, genetic fusion, non-covalent association orotherwise, to one or more other molecular entities. For example, theanti-PSMA antibody, or antigen-binding fragment thereof, can be coupledto a label, such as a fluorescent label, a biologically active enzymelabel, a radioisotope (e.g., a radioactive ion), a nuclear magneticresonance active label, a luminescent label, or a chromophore. In otherembodiments, the anti-PSMA antibody, or antigen-binding fragmentthereof, can be coupled to a therapeutic agent, e.g., a cytotoxicmoiety, e.g., a therapeutic drug, a radioisotope, molecules of plant,fungal, or bacterial origin, or biological proteins (e.g., proteintoxins) or particles (e.g., recombinant viral particles, e.g., via aviral coat protein), or mixtures thereof. The therapeutic agent can bean intracellularly active drug or other agent, such as short-rangeradiation emitters, including, for example, short-range, high-energyα-emitters, as described herein. In some preferred embodiments, theanti-PSMA antibody, or antigen binding fragment thereof, can be coupledto a molecule of plant or bacterial origin (or derivative thereof),e.g., a maytansinoid (e.g., maytansinol or the DM1 maytansinoid, seeFIG. 15), a taxane, or a calicheamicin. A radioisotope can be an α-, β-,or γ-emitter, or an β- and γ-emitter. Radioisotopes useful astherapeutic agents include yttrium (⁹⁰Y), lutetium (¹⁷⁷Lu), actinium(²²⁵Ac), praseodymium, astatine (²¹¹At), rhenium (¹⁸⁶Re), bismuth (²¹²Bior ²¹³Bi), and rhodium (¹⁸⁸Rh). Radioisotopes useful as labels, e.g.,for use in diagnostics, include iodine (¹³¹I or ¹²⁵I), indium (¹¹¹In),technetium (⁹⁹mTc), phosphorus (³²P), carbon (¹⁴C), and tritium (³H), orone of the therapeutic isotopes listed above. The anti-PSMA antibody, orantigen-binding fragment thereof can also be linked to another antibodyto form, e.g., a bispecific or a multispecific antibody.

In another aspect, the invention features, an anti-PSMA antibody, e.g.,an antibody described herein, coupled, e.g., by covalent linkage, to aproteosome inhibitor or a topoisomerase inhibitor.[(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(3-mercaptoacetyl)amino]propyl]amino]butyl]Boronic acid is a suitable proteosome inhibitor.N,N′-bis[2-(9-methylphenazine-1-carboxamido)ethyl]-1,2-ethanediamine isa suitable topoisomerase inhibitor.

In some embodiments, the anti-PSMA antibody is linked to a therapeuticagent as described herein via a linker, e.g., a cleavable linker, e.g.,a cleavable linker that allows the release of the therapeutic agent intothe intracellular space upon internalization of the antibody-agentcomplex.

In another aspect, the invention provides compositions, e.g.,pharmaceutical compositions, which include a pharmaceutically acceptablecarrier, excipient or stabilizer, and at least one of the anti-PSMAantibodies, e.g., the modified anti-PSMA antibodies (or fragmentsthereof) described herein. In a preferred embodiment, the pharmaceuticalcomposition includes about 2-10 mg/ml, preferably about 4-6 mg/ml, andmore preferably about 5.0±0.5 mg/ml of a conjugated or unconjugated(naked) antibody described herein. (When a conjugated antibody is used,the mg/ml used preferably refers to the milligrams of antibody, asopposed to the milligrams of conjugated drug). The composition canfurther include one or more of: sodium succinate, e.g., about 10 mM toabout 30 mM, preferably about 20 mM, NaSuccinate (C₄H₄O₄Na₂), andsucrose, e.g., about 75-125, preferably 100, mg/ml sucrose. In apreferred embodiment, the composition includes both sodium succinate andsucrose. A preferred composition includes about 10 mM to about 30 mM,preferably about 20 mM, NaSuccinate and about 75 to 125 mg/ml,preferably about 100 mg/ml, sucrose. A preferred pH of the sodiumsuccinate is about 4-6.5, preferably 5.5. In a preferred embodiment theantibody is conjugated to a label or a therapeutic agent. In oneembodiment, the compositions, e.g., the pharmaceutical compositions,comprise a combination of two or more of the aforesaid anti-PSMAantibodies. For example, a composition, e.g., pharmaceuticalcomposition, which comprises a deimmunized J591 antibody, in combinationwith another anti-PSMA antibody, or an antibody to another tumorcell-associated antigen, e.g., EGF receptor, Her-2/neu, etc.Combinations of the anti-PSMA antibody (e.g., naked or conjugated) and adrug, e.g., a therapeutic agent (e.g., a cytotoxic or cytostatic drug,e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxy anthracin dione, estramustine,mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol,puromycin, maytansinoids, e.g., DM1, calicheamicin, or taxanes, e.g.,TAXOL® (paclitaxel), paclitaxel, and/or docetaxel, topoisomeraseinhibitors, or an immunomodulatory agent, e.g., IL-1, 2, 4, 6, or 12,interferon alpha or gamma, or immune cell growth factors such as GCSF orGM-CSF) are also within the scope of the invention. The pharmaceuticalcompositions of the invention can be stored frozen, e.g., below zerodegrees Celsius, preferably at about −4° C., more preferably at about−20° C., even more preferably at about −70 to −90° C.

In a preferred embodiment, the pharmaceutical composition comprises lessthan about 20%, less than about 15%, less than about 10%, less thanabout 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% impurities, e.g.,protein or non-protein impurities. The presence and percentage ofimpurities in a sample, e.g., a sample from a batch of thepharmaceutical composition, can be determined by any method known in theart, including but not limited to spectrophotometry, IEF, SEC, SDS-PAGE,LC, CE and/or MS. The sample can be analyzed under native conditions orafter application of a treatment, e.g., reduction and/or denaturation.Typical impurities include small molecule impurities, e.g., impuritiesarising from the manufacturing process, e.g., the manufacturing processof a conjugated antibody. Examples of such impurities includeunconjugated conjugate (e.g., DM1, DM1 dimer, or DM1-TPA in apharmaceutical composition which also includes DM1-conjugatedantibodies), TPA, EDTA, N-succinimidyl 4-(2-pyridyldithiopentanoate),2-pyridyldithiopentanoate, and dimethylacetamide. Other impurities caninclude protein-related substances, e.g., antibody dimers and improperlyconjugated antibodies in a pharmaceutical composition comprisingconjugated antibodies. The pharmaceutical composition can comprisegreater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%conjugated or unconjugated antibody monomer.

In some embodiments, the pharmaceutical composition includes ananti-PSMA antibody (or fragment thereof) linked to a therapeutic agent,e.g., a cytotoxic agent such as DM1. The ratio of antibody totherapeutic agent (e.g., DM1) can be determined by any method known inthe art, including but not limited to spectrophotometry, IEF, SEC,SDS-PAGE, LC, CE and/or MS. For example, the therapeutic agent/antibodyratio can be determined by measuring the total conjugate molarconcentration in a sample, e.g., spectrophotometrically, and dividing bythe molar concentration of the conjugated antibody. Where the conjugateis DM1, the DM¹/antibody ratio is preferably about 2.0-5.0, morepreferably about 3.0-4.0, even more preferably about 3.4 to 3.8 (e.g.,3.5).

The invention also features nucleic acid sequences that encode a heavyand light chain immunoglobulin described herein. For example, theinvention features, a first and second nucleic acid encoding a modifiedheavy and light chain variable region, respectively, of a modifiedanti-PSMA antibody molecule as described herein. In another aspect, theinvention features host cells and vectors containing the nucleic acidsof the invention.

In another aspect, the invention features a method of producing ananti-PSMA antibody, e.g., a modified anti-PSMA antibody, orantigen-binding fragment thereof. The method includes:

-   -   providing a first nucleic acid encoding a heavy chain variable        region, e.g., a modified heavy chain variable region as        described herein;    -   providing a second nucleic acid encoding a light chain variable        region, e.g., a modified light chain variable region as        described herein; and    -   introducing said first and second nucleic acids into a host cell        under conditions that allow expression and assembly of said        light and heavy chain variable regions.

The first and second nucleic acids can be linked or unlinked, e.g.,expressed on the same or different vector, respectively.

The host cell can be a eukaryotic cell, e.g., a mammalian cell, aninsect cell, a yeast cell, or a prokaryotic cell, e.g., E. coli. Forexample, the mammalian cell can be a cultured cell or a cell line.Exemplary mammalian cells include lymphocytic cell lines (e.g., NS0),Chinese hamster ovary cells (CHO), COS cells, oocyte cells, and cellsfrom a transgenic animal, e.g., mammary epithelial cell. For example,nucleic acids encoding the modified antibody described herein can beexpressed in a transgenic animal. In one embodiment, the nucleic acidsare placed under the control of a tissue-specific promoter (e.g., amammary specific promoter) and the antibody is produced in thetransgenic animal. For example, the antibody molecule is secreted intothe milk of the transgenic animal, such as a transgenic cow, pig, horse,sheep, goat or rodent.

The invention also features a method of ablating or killing, a cell,e.g., a prostatic cell (e.g., a cancerous or non-cancerous prostaticcell, e.g., a normal, benign or hyperplastic prostatic epithelial cell),or a malignant, non-prostatic cell, e.g., cell found in a non-prostaticsolid tumor that, e.g., has vasculature which expresses PSMA, a softtissue tumor, or a metastatic lesion (e.g., a cell found in renal,urothelial (e.g. bladder), colonic, rectal, pulmonary, breast or hepaticcancers and/or metastases thereof). Methods of the invention includecontacting the cell, or a nearby cell, e.g., a vascular endothelial cellproximate to the cell, with an anti-PSMA antibody as described herein,e.g., a modified anti-PSMA antibody, in an amount sufficient to ablateor kill, the cell. Alternatively, an anti-PSMA antibody as describedherein, e.g., a modified anti-PSMA antibody, preferably a fragment of amodified anti-PSMA antibody, can be conjugated to a viral particle,e.g., to a coat protein of a viral particle. The anti-PSMA/viralparticle conjugate can be used to target prostate cells, e.g., cancerousprostate cells, with genetically engineered viral particles that infectthe cells and express, e.g., pro apoptotic genes, to thereby kill thecells or inhibit cell growth.

The methods can be used on cells in culture, e.g. in vitro or ex vivo.For example, prostatic cells (e.g., malignant or normal, benign orhyperplastic prostate epithelial cells) or non-prostatic cancerous ormetastatic cells (e.g., renal, urothelial (e.g., bladder), testicular,colon, rectal, lung (e.g., non-small cell lung carcinoma), breast,liver, neural (e.g., neuroendocrine), glial (e.g., glioblastoma),pancreatic (e.g., pancreatic duct), melanoma (e.g., malignant melanoma),or soft tissue sarcoma cancerous cells) can be cultured in vitro inculture medium and the contacting step can be effected by adding themodified anti-PSMA antibody or fragment thereof, to the culture medium.The method can be performed on cells (e.g., prostatic cells, ornon-prostatic cancerous or metastatic cells) present in a subject, aspart of an in vivo (e.g., therapeutic or prophylactic) protocol.

Methods of the invention can be used, for example, to treat or prevent adisorder, e.g., a prostatic disorder (e.g., a cancerous or non-cancerousprostatic disorder, e.g., a benign or hyperplastic prostatic disorder),or a non-prostatic disorder (e.g., cancer, e.g., malignant cancer), byadministering to a subject an antibody described herein, preferably amodified PSMA antibody, or antigen-binding fragment thereof, in anamount effective to treat or prevent such disorder. Particularlypreferred antibodies include modified antibodies having CDRs from any ofa J591, J415, J533 or E99, and in particular deimmunized versions ofthese antibodies, particularly deJ591 or deJ415. Examples of prostaticdisorders that can be treated or prevented include, but are not limitedto, genitourinary inflammation (e.g., inflammation of smooth musclecells) as in prostatitis; benign enlargement, for example, nodularhyperplasia (benign prostatic hypertrophy or hyperplasia); and cancer,e.g., adenocarcinoma or carcinoma, of the prostate and/or testiculartumors. Methods and compositions disclosed herein are particularlyuseful for treating metastatic lesions associated with prostate cancer.In some embodiments, the patient will have undergone one or more ofprostatectomy, chemotherapy, or other anti-tumor therapy and the primaryor sole target will be metastatic lesions, e.g., metastases in the bonemarrow or lymph nodes. Examples of non-prostatic cancerous disordersinclude, but are not limited to, solid tumors, soft tissue tumors,liquid tumors and particularly metastatic lesions. Examples of solidtumors include malignancies, e.g., sarcomas, adenocarcinomas, andcarcinomas, of the various organ systems, such as those affecting lung,breast, lymphoid, gastrointestinal (e.g., colon), genitals andgenitourinary tract (e.g., renal, urothelial, bladder cells), pharynx,CNS (e.g., neural or glial cells), skin (e.g., melanoma), and pancreas,as well as adenocarcinomas which include malignancies such as most coloncancers, rectal cancer, renal-cell carcinoma, liver cancer, non-smallcell carcinoma of the lung, cancer of the small intestine and cancer ofthe esophagus. Methods and compositions disclosed herein areparticularly useful for treating metastatic lesions associated with theaforementioned cancers. In some embodiments, the patient will haveundergone one or more of surgical removal of a tissue, chemotherapy, orother anticancer therapy and the primary or sole target will bemetastatic lesions, e.g., metastases in the bone marrow or lymph nodes.

In a preferred embodiment the subject is treated to prevent a disorder,e.g., a prostatic disorder. The subject can be one at risk for thedisorder, e.g., a subject having a relative afflicted with the disorder,e.g., a subject with one or more of a grandparent, parent, uncle oraunt, sibling, or child who has or had the disorder, or a subject havinga genetic trait associated with risk for the disorder. In a preferredembodiment the disorder is a prostatic disorder (e.g., a cancerous ornon-cancerous prostatic disorder, e.g., a benign or hyperplasticprostatic disorder), or a non-prostatic disorder (e.g., cancer, e.g.,malignant cancer) and the subject has one or more of a grandfather,father, uncle, brother, or son who has or had the disorder, or a subjecthaving a genetic trait associated with risk for the disorder.

The subject can be a mammal, e.g., a primate, preferably a higherprimate, e.g., a human (e.g., a patient having, or at risk of, adisorder described herein, e.g., a prostatic or a cancer disorder). Inone embodiment, the subject is a patient having prostate cancer (e.g., apatient suffering from recurrent or metastatic prostate cancer).

The anti-PSMA antibody or fragment thereof, e.g., a modified anti-PSMAantibody or fragment thereof as described herein, can be administered tothe subject systemically (e.g., orally, parenterally, subcutaneously,intravenously, rectally, intramuscularly, intraperitoneally,intranasally, transdermally, or by inhalation or intracavitaryinstallation), topically, or by application to mucous membranes, such asthe nose, throat and bronchial tubes.

The methods of the invention, e.g., methods of treatment or preventing,can further include the step of monitoring the subject, e.g., for achange (e.g., an increase or decrease) in one or more of: tumor size;levels of a cancer marker, e.g., levels of PSA, alkaline phosphatase, orserum hemoglobin for a patient with prostate cancer; the rate ofappearance of new lesions, e.g., in a bone scan; the appearance of newdisease-related symptoms; the size of soft tissue mass, e.g., adecreased or stabilization; quality of life, e.g., amount of diseaseassociated pain, e.g., bone pain; or any other parameter related toclinical outcome. The subject can be monitored in one or more of thefollowing periods: prior to beginning of treatment; during thetreatment; or after one or more elements of the treatment have beenadministered. Monitoring can be used to evaluate the need for furthertreatment with the same modified anti-PSMA antibody or fragment thereofor for additional treatment with additional agents. Generally, adecrease in one or more of the parameters described above is indicativeof the improved condition of the subject, although with serum hemoglobinlevels, an increase can be associated with the improved condition of thesubject.

The methods of the invention can further include the step of analyzing anucleic acid or protein from the subject, e.g., analyzing the genotypeof the subject. In one embodiment, a nucleic acid encoding human PSMAand/or an upstream or downstream component(s) of human PSMA signalling,e.g., an extracellular or intracellular activator or inhibitor of humanPSMA, is analyzed. The analysis can be used, e.g., to evaluate thesuitability of, or to choose between alternative treatments, e.g., aparticular dosage, mode of delivery, time of delivery, inclusion ofadjunctive therapy, e.g., administration in combination with a secondagent, or generally to determine the subject's probable drug responsephenotype or genotype. The nucleic acid or protein can be analyzed atany stage of treatment, but preferably, prior to administration of themodified anti-PSMA antibody or fragment thereof to thereby determineappropriate dosage(s) and treatment regimen(s) of the modified anti-PSMAantibody or fragment thereof (e.g., amount per treatment or frequency oftreatments) for prophylactic or therapeutic treatment of the subject.

The anti-PSMA antibody or fragment thereof (e.g., a modified anti-PSMAantibody or fragment thereof described herein) can be used alone inunconjugated form to thereby ablate or kill the PSMA-expressingprostatic or cancerous cells by, e.g., antibody-dependent cell killingmechanisms such as complement-mediated cell lysis and/or effectorcell-mediated cell killing. In other embodiments, the anti-PSMA antibodyor fragment thereof (e.g., a modified anti-PSMA antibody or fragmentthereof described herein) can be bound to a substance, e.g., a cytotoxicagent or moiety, e.g., a therapeutic drug, a compound emittingradiation, molecules of plant, fungal, or bacterial origin, or abiological protein (e.g., a protein toxin) or particle (e.g., arecombinant viral particle, e.g., via a viral coat protein). Forexample, the anti-PSMA antibody, or antigen-binding fragment thereof,can be coupled to a radioactive isotope such as an α-, β-, or γ-emitter,or a β- and γ-emitter. Examples of radioactive isotopes include iodine(¹³¹I or ¹²⁵I), yttrium (⁹⁰Y), lutetium (¹⁷⁷Lu), actinium (²²⁵Ac),praseodymium, or bismuth (²¹²Bi or ²¹³Bi). Alternatively, the anti-PSMAantibody, or antigen-binding fragment thereof, can be coupled to abiological protein, a molecule of plant or bacterial origin (orderivative thereof), e.g., a maytansinoid (e.g., maytansinol or DM1), aswell as a taxane (e.g., TAXOL® (paclitaxel) or taxotere), orcalicheamicin. The maytansinoid can be, for example, maytansinol or amaytansinol analogue. Examples of maytansinol analogues include thosehaving a modified aromatic ring (e.g., C-19-dechloro, C-20-demethoxy,C-20-acyloxy) and those having modifications at other positions (e.g.,C-9-CH, C-14-alkoxymethyl, C-14-hydroxymethyl or aceloxymethyl,C-15-hydroxy/acyloxy, C-15-methoxy, C-18-N-demethyl, 4,5-deoxy).Maytansinol and maytansinol analogues are described, for example, inU.S. Pat. No. 6,333,410, the contents of which are incorporated hereinby reference. The calicheamicin can be, for example, a bromo-complexcalicheamicin (e.g., an alpha, beta or gamma bromo-complex), aniodo-complex calicheamicin (e.g., an alpha, beta or gamma iodo-complex),or analogs and mimics thereof. Bromo-complex calicheamicins includeα₁-BR, α₂-BR, α₃-BR, α₄-BR, β₁-BR, β₂-BR and γ₁-BR. Iodo-complexcalicheamicins include α₁-I, α₂-I, α₃-I, β₂-I, δ₁-I and γ₁-BR.Calicheamicin and mutants, analogs and mimics thereof are described, forexample, in U.S. Pat. No. 4,970,198, issued Nov. 13, 1990, U.S. Pat. No.5,264,586, issued Nov. 23, 1993, U.S. Pat. No. 5,550,246, issued Aug.27, 1996, U.S. Pat. No. 5,712,374, issued Jan. 27, 1998, and U.S. Pat.No. 5,714,586, issued Feb. 3, 1998, the contents of which areincorporated herein by reference. Maytansinol can be coupled toantibodies using, e.g., an N-succinimidyl 3-(2-pyridyldithio)proprionate(also known as N-succinimidyl 4-(2-pyridyldithio)pentanoate or SPP),4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio)-toluene (SMPT),N-succinimidyl-3-(2-pyridyldithio)butyrate (SDPB), 2-iminothiolane, orS-acetyl succinic anhydride.

The antibodies described herein (e.g., a human antibody or modifiedantibody described herein) can be administered to a subject in single ormultiple doses to treat or prevent a prostatic or cancerous disorder,e.g., a prostatic or cancerous disorder described herein. In oneembodiment, the methods of the invention include administering to thesubject two or more doses of an antibody molecule described hereincoupled to lutetium (¹⁷⁷Lu), wherein each dose is about 40 to 65%,preferably about 40% to 60%, 45% to 55% of the maximum tolerated dose(MTD) of the antibody molecule coupled to lutetium (¹⁷⁷Lu). The antibodycoupled to ¹⁷⁷Lu can be given in two, three, four, five, six, seven,eight, nine or ten doses, e.g., over a period of a dose once every week,two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks,eight weeks, or more. In a preferred embodiment, the subject isadministered up to three, four or five doses, e.g., with a doseadministered once every four to eight weeks. Each dose can be at aboutthe same amount as the other doses or one or more doses can differ fromeach other but it is preferred that no dose given is greater than 65% ofthe MTD of the antibody molecule coupled to ¹⁷⁷Lu. In one embodiment,the method of treating or preventing a prostatic or cancerous disorderincludes administering to the subject two or more doses of a deimmunizedJ591, e.g., a deimmunized J591 as described herein, coupled to ¹⁷⁷Lu,wherein each dose is administered at less than 60 mCi/m². Preferably,each dose of the deimmunized J591 antibody molecule coupled to ¹⁷⁷Lu isadministered at less than 45 mCi/m², e.g., 30 mCi/m², 15 mCi/m² or less.

When multiple doses are planned or administered, the methods can furtherinclude evaluating the subject after one or more of the doses forhematologic toxicity and/or non-hematologic toxicity. Hematologictoxicity can be evaluated by analyzing the subject for myelosuppressionsuch as thrombocytopenia, granulocytopenia or both. The presence ofnon-hematologic toxicity can be determined by analyzing the subject forthe presence and severity of one or more of: fatigue, anorexia, fever,rigors, nausea, vomiting, diarrhea, constipation, ALT levels and ASTlevels. The methods can further include making a determination ofwhether an additional dose or doses of the antibody coupled to ¹⁷⁷Luwill be administered to the subject. Such a determination can be basedupon the determined level of hematologic toxicity (e.g.,myelosuppression) and/or non-hematologic toxicity in the subject afteradministration of one or more of the multiple doses of the antibodymolecule coupled to lutetium (¹⁷⁷Lu). For example, the decision toadminister an additional dose or doses of the antibody can be based upona finding that the hematologic toxicity is less than grade 4thrombocytopenia and/or less than grade 4 granulocytopenia (e.g.,neutropenia) for at least 5, 6, 7, 8, 10 or more days. In addition,based upon the evaluation of hematologic toxicity and/or non-hematologictoxicity, the amount of a subsequent dose or doses can be adjustedaccording to the subject's hematologic and/or non-hematologic responseto the previous dose or doses. For example, a decision can be made toadminister a subsequent dose or doses at about 40 to 60% of the MTD ofthe antibody molecule coupled to ¹⁷⁷Lu or a decision can be made toadminister the subsequent dose or doses in an amount less than 40%, 35%,30%, 25%, 20%, 15% of the MTD. The evaluation of hematologic toxicityand/or non-hematologic toxicity can also be used to determine whether atherapeutic modality that enhances blood cell counts should beadministered to the subject. For example, a therapeutic modality thatenhances blood counts can be administered prior to, in conjunction with,or after a subsequent dose (or doses) of the antibody is administered tothe subject. Examples of therapeutic modalities that enhance bloodcounts include: platelet transfusion, administration of a growth factor(e.g., thrombopoietin, epoietin α, erythropoietin, G-CSF, GM-CSF, andinterleukins (e.g., IL-11)) and bone marrow transplantation.

Methods of the invention for treating or preventing a prostatic orcancerous disorder by administering multiple doses of an antibodymolecule described herein coupled to ¹⁷⁷Lu can include a step ofselecting a subject which is less likely to exhibit hematologic toxicityafter one or more doses of the antibody molecule. For example, bloodcounts (e.g., platelet and/or granulocyte levels) of the subject priorto treatment can be used to select subjects less likely to exhibithematologic toxicity after one or more doses. Subjects having normalblood counts or subjects having low blood counts but treated with atherapeutic modality which enhances blood counts prior to administrationof the antibody molecule can be selected. Normal levels of platelet andgranulocytes (e.g., neutrophils, eosinophils and basophils) are known.For example, normal platelet counts are normally about 140,000/μL to440,000/μL. Platelet counts below these levels, e.g., platelet countsbelow 100,000/μL, 50,000/μL or less are considered low, while plateletcounts below 10,000/μL indicates severe thrombocytopenia. Neutrophilsare normally present at about 2,500 cell/mm² to 6000 cells/mm².Neutrophil levels below these levels, e.g., neutrophil counts belowthese levels, e.g., below 2,000 cells/mm², 1,500 cells/mm², 1,000cells/mm² or less, are considered low. Examples of therapeuticmodalities which enhance blood counts are described herein.

In yet another embodiment, the antibody molecules of the invention(e.g., the modified antibody molecules described herein) can be coupledto ¹⁷⁷Lu and administered in multiple doses to a subject such thatcumulative radiation in the subject is less than 315 mCi/m², 270 mCi/m²,225 mCi/m² over a period of about 1 year, 9 months, 8 months, 7 months,6 months or less, to thereby treat or prevent a prostatic or cancerousdisorder. Preferably, multiple doses of the antibody coupled to 177 Luis administered such that cumulative radiation is 210 mCi/m², 180mCi/m², 150 mCi/m² or less over a period of less than 8 months, 7months, 6 months, 5 months, 4 months or 3 months.

In another aspect, the invention features, a method of making adecision, e.g., a medical or financial decision. The method includes:generating or receiving data regarding hematologic and/ornon-hematologic toxicity of a subject who has received one, two, three,four, five or more doses of an antibody described herein (e.g., a humanor modified antibody described herein, e.g., a human or modifiedantibody described herein coupled to ¹⁷⁷Lu), e.g., data generated by amethod described herein; and using the data to make the decision, e.g.,selecting between a first outcome and a second outcome. In a preferredembodiment, the data is an indicator of whether a subject is amendableto an additional dose or doses of the antibody. In a preferredembodiment, the decision is made by comparing the data to a referencestandard and making the decision based on the relationship of the datato the reference. For example, the data can be a value or other term forthe likelihood of a subject developing hematologic and/ornon-hematologic toxicity upon further dosing with the antibody and ifthe value or other term has a preselected relationship to the referencestandard, e.g., if the value or term in the data is less than areference standard, selecting a first outcome and if the data is greaterthan a reference standard selecting a second outcome. An outcome can beproviding or not providing service or treatment (e.g., providing or notproviding further dosing) or paying for or not paying for all or part ofa service or treatment or paying or not paying for further treatment(e.g., subsequent dosing with the antibody). In a preferred embodiment,the first outcome is suggesting or providing a first course of medicaltreatment, e.g., any treatment described herein, and the second courseis suggesting that the treatment not be given or not providing thetreatment.

In a preferred embodiment the first outcome includes or results in theauthorization or transfer of funds to pay for a service or treatmentprovided to a subject and the second outcome includes or results in therefusal to pay for a service or treatment provided to a subject. Forexample, an entity, e.g., a hospital, care giver, government entity, oran insurance company or other entity which pays for, or reimbursesmedical expenses, can use the outcome of a method described herein todetermine whether a party, e.g., a party other than the subject patient,will pay for services or treatment provided to the patient. For example,a first entity, e.g., an insurance company, can use the outcome of amethod described herein to determine whether to provide financialpayment to, or on behalf of, a patient, e.g., whether to reimburse athird party, e.g., a vendor of goods or services, a hospital, physician,or other care-giver, for a service or treatment provided to a patient.For example, a first entity, e.g., an insurance company, can use theoutcome of a method described herein to determine whether to continue,discontinue, enroll an individual in an insurance plan or program, e.g.,a health insurance or life insurance plan or program.

The methods and compositions of the invention can be used in combinationwith other therapeutic modalities. In one embodiment, the methods of theinvention include administering to the subject a modified anti-PSMAantibody or fragment thereof, e.g., a modified anti-PSMA antibody orfragment thereof as described herein, in combination with a cytotoxicagent, in an amount effective to treat or prevent said disorder. Theantibody and the cytotoxic agent can be administered simultaneously orsequentially. In other embodiments, the methods and compositions of theinvention are used in combination with surgical and/or radiationprocedures. In yet other embodiments, the methods can be used incombination with immunomodulatory agents, e.g., IL-1, 2, 4, 6, or 12, orinterferon alpha or gamma, or immune cell growth factors such as GCSFand/or GM-CSF.

Exemplary cytotoxic agents that can be administered in combination withthe anti-PSMA antibodies (naked or conjugated) include antimicrotubuleagents, topoisomerase inhibitors, antimetabolites, mitotic inhibitors,alkylating agents, intercalating agents, agents capable of interferingwith a signal transduction pathway, agents that promote apoptosis andradiation. In one embodiment, the cytotoxic agent that can beadministered with an anti-PSMA antibody described herein is TAXOL®(paclitaxel), cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D,I-dihydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids,e.g., maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat.Nos. 5,475,092, 5,585,499, 5,846,545) and/or analogs or homologsthereof.

In therapies of prostatic disorders, e.g., prostate cancer, theanti-PSMA antibodies can be used in combination with existingtherapeutic modalities, e.g., prostatectomy (partial or radical),radiation therapy, hormonal therapy, androgen ablation therapy, andcytotoxic chemotherapy. Typically, hormonal therapy works to reduce thelevels of androgens in a patient, and can involve administering aleuteinizing hormone-releasing hormone (LHRH) analog or agonist (e.g.,Lupron, Zoladex, leuprolide, buserelin, or goserelin), as well asantagonists (e.g., Abarelix). Non-steroidal anti-androgens, e.g.,flutamide, bicalutimade, or nilutamide, can also be used in hormonaltherapy, as well as steroidal anti-androgens (e.g., cyproterone acetateor megastrol acetate), estrogens (e.g., diethylstilbestrol), surgicalcastration, PROSCAR™, secondary or tertiary hormonal manipulations(e.g., involving corticosteroids (e.g., hydrocortisone, prednisone, ordexamethasone), ketoconazole, and/or aminogluthethimide), inhibitors of5a-reductase (e.g., finisteride), herbal preparations (e.g., PC-SPES),hypophysectomy, and adrenalectomy. Furthermore, hormonal therapy can beperformed intermittently or using combinations of any of the abovetreatments, e.g., combined use of leuprolide and flutamide. Theanti-PSMA antibodies described herein can be used in combination withanother antibody, e.g., another antibody that binds to PSMA or anantigen other than PSMA, e.g., another antigen expressed on prostatecancer cells. One or both of the anti-PSMA antibody can be conjugated orunconjugated. When both are conjugated, they can be conjugated with thesame or different therapeutic agents or labels. In one embodiment, theanti-PSMA antibody can be administered with at least one or moreadditional antibodies.

Any combination and sequence of anti-PSMA antibodies (e.g., a modifiedanti-PSMA antibody or fragment thereof described herein) and othertherapeutic modalities can be used. The anti-PSMA antibody and othertherapeutic modalities can be administered during periods of activedisorder, or during a period of remission or less active disease. Theanti-PSMA and other therapeutic modalities can be administered beforetreatment, concurrently with treatment, posttreatment, or duringremission of the disorder.

In another aspect, the invention features methods for detecting thepresence of a PSMA protein in a sample in vitro (e.g., a biologicalsample, e.g., serum, semen or urine, or a tissue biopsy, e.g., from aprostatic or cancerous lesion). The subject method can be used toevaluate, e.g., diagnose or stage a disorder described herein, e.g., aprostatic or cancerous disorder. The method includes: (i) contacting thesample (and optionally, a reference, e.g., a control sample) with ananti-PSMA antibody, or fragment thereof, e.g., a modified anti-PSMAantibody or fragment thereof as described herein, under conditions thatallow interaction of the anti-PSMA antibody and the PSMA protein tooccur; and (ii) detecting formation of a complex between the anti-PSMAantibody, and the sample (and optionally, the reference, e.g., control,sample). Formation of the complex is indicative of the presence of PSMAprotein, and can indicate the suitability or need for a treatmentdescribed herein. For example, a statistically significant change in theformation of the complex in the sample relative to the reference sample,e.g., the control sample, is indicative of the presence of PSMA in thesample. In some embodiments, the methods can include the use of morethan one anti-PSMA antibody, e.g., two anti-PSMA antibodies that bind todifferent epitopes on PSMA. For example, the method can involve an ELISAassay, e.g., as described in Example 21. In some embodiments, the methodcan be used to select a subject for administration of a composition asdescribed herein, e.g., a composition comprising an anti-PSMA antibody(or fragment thereof) coupled to a therapeutic agent, to treat thesubject. For example, if the presence of PSMA is detected in a samplederived from a subject, that subject can then be selected foradministration of a modified anti-PSMA antibody, e.g., an antibodydescribed herein.

In yet another aspect, the invention provides a method for detecting thepresence of PSMA in vivo (e.g., in vivo imaging in a subject). Themethod can be used to evaluate, e.g., diagnose or stage a disorderdescribed herein, e.g., a prostatic or a cancerous disorder, in asubject, e.g., a mammal, e.g., a primate, e.g., a human. The methodincludes: (i) administering to a subject an anti-PSMA antibody orantigen binding fragment thereof (e.g., a modified anti-PSMA antibody orfragment thereof described herein), under conditions that allowinteraction of the anti-PSMA antibody (or fragment thereof) and the PSMAprotein to occur; and (ii) detecting formation of a complex between theantibody or fragment and PSMA. A statistically significant change in theformation of the complex in the subject relative to the reference, e.g.,the control subject or subject's baseline, is indicative of the presenceof the PSMA. In some embodiments, the method can be used to select asubject for administration of a composition as described herein, e.g., acomposition comprising an anti-PSMA antibody (or fragment thereof),e.g., an anti-PSMA antibody described herein, coupled to a therapeuticagent, to treat the subject. For example, if the presence of PSMA isdetected in a sample derived from a subject, that subject can then beselected for administration of an anti-PSMA antibody (e.g., a modifiedanti-PSMA antibody described herein).

In other embodiments, a method of diagnosing or staging a disorder asdescribed herein (e.g., a prostatic or cancerous disorder) is provided.The method includes: (i) identifying a subject having, or at risk ofhaving, the disorder; (ii) obtaining a sample of a tissue or cellaffected with the disorder; (iii) contacting said sample or a controlsample with an anti-PSMA antibody as described herein, e.g., a modifiedanti-PSMA antibody or fragment, under conditions that allow interactionof the binding agent and the PSMA protein to occur, and (iv) detectingformation of a complex. A statistically significant increase in theformation of the complex between the antibody (or fragment thereof) withrespect to a reference sample, e.g., a control sample, is indicative ofthe disorder or the stage of the disorder. In some embodiments, themethod can be used to select a subject for administration of acomposition as described herein, e.g., a composition comprising ananti-PSMA antibody (or fragment thereof) described herein, to treat thesubject. For example, if the presence of PSMA is detected in a samplederived from a subject, that subject can then be selected foradministration of a modified anti-PSMA antibody.

Preferably, the anti-PSMA antibody or fragment thereof, used in the invivo and in vitro diagnostic methods is directly or indirectly labeledwith a detectable substance to facilitate detection of the bound orunbound binding agent. Suitable detectable substances include variousbiologically active enzymes, prosthetic groups, fluorescent materials,luminescent materials, paramagnetic (e.g., nuclear magnetic resonanceactive) materials, and radioactive materials. In some embodiments, theanti-PSMA antibody or fragment thereof is coupled to a radioactive ion,e.g., indium (¹¹¹In), iodine (¹³¹I or ¹²⁵I) yttrium (⁹⁰Y), lutetium(¹⁷⁷Lu), actinium (²²⁵Ac), bismuth (²¹²Bi or ²¹³Bi), sulfur (³⁵S),carbon (¹⁴C), tritium (³H), rhodium (¹⁸⁸Rh) technetium (⁹⁹mTc),praseodymium, or phosphorous (³²P).

In another aspect, the invention provides a method for determining thedose, e.g., radiation dose, that different tissues are exposed to when asubject, e.g., a human subject, is administered an anti-PSMA antibodythat is conjugated to a radioactive isotope. The method includes: (i)administering an anti-PSMA antibody as described herein, e.g., amodified anti-PSMA antibody, that is labeled with a radioactive isotope,e.g., ¹¹¹In, to a subject; (ii) measuring the amount of radioactiveisotope located in different tissues, e.g., prostate, liver, kidney, orblood, at various time points until most, e.g., 50%, 80%, 90%, 95%, ormore, of the radioactive isotope has been eliminated from the body ofthe subject; and (iii) calculating the total dose of radiation receivedby each tissue analyzed. In some embodiments, the measurements are takenat scheduled time points, e.g., day 1, 2, 3, 5, 7, and 12 or day 2, 4, 6and 14, following administration (at day 0) of the radioactively labeledanti-PSMA antibody to the subject. In some embodiments, the radiationdose that a tissue receives for one radioactive isotope, e.g., agamma-emitter, e.g., ¹¹¹In, can be used to calculate the expected dosethat the same tissue would receive from a different radioactive isotope,e.g., a beta-emitter, e.g., ⁹⁰Y.

In another aspect, the invention features methods of treating pain,e.g., reducing pain, experienced by a subject having or diagnosed withprostate disease, e.g., benign prostatic hyperplasia or prostate cancer,or non-prostate cancer, e.g., a cancer having vasculature whichexpresses PSMA (e.g., renal, urothelial (e.g., bladder), testicular,colon, rectal, lung (e.g., non-small cell lung carcinoma), breast,liver, neural (e.g., neuroendocrine), glial (e.g., glioblastoma), orpancreatic (e.g., pancreatic duct) cancer, melanoma (e.g., malignantmelanoma), or soft tissue sarcoma). The methods include administering ananti-PSMA antibody as described herein, e.g., a modified anti-PSMAantibody, to a subject in an amount sufficient to treat, e.g., reduce,the pain associated with prostate disease or non-prostate cancer. Insome embodiments, the subject may have no signs of prostate disease ornon-prostate cancer other than, e.g., elevated levels of serum PSA andthe sensation of pain. The pain can be bone pain, as well as, painassociated with obstructive voiding symptoms due to enlarged prostate,e.g., urinary hesitancy or diminished urinary stream, frequency ornocturia. The treatment of pain using the modified anti-PSMA antibodiesof the invention can lead to a decreased or dramatically lowered need,or even eliminate the need, for analgesics, e.g., narcotics. Inaddition, by reducing pain, the methods of treatment can restore themobility of, e.g., limbs, that have become dysfunctional as a result ofpain associated with movement.

In some embodiments, the modified anti-PSMA antibody is administered inan unconjugated form in an amount sufficient to treat, e.g., reduce,pain associated with prostate disease or non-prostate cancer. In otherembodiments, the modified anti-PSMA antibody, or antigen-bindingfragment thereof, is administered in a derivatized form, e.g., linked toanother functional molecule, as described herein.

The method of treating pain experienced by a subject having or diagnosedwith benign prostatic hyperplasia or prostate cancer, or non-prostatecancer, can include, for example, administering two or more doses of anantibody or antigen binding fragment thereof as described herein coupledto lutetium (¹⁷⁷Lu). Each dose can be about 40 to 65% of the maximumtolerated dose (MTD) of the antibody molecule coupled to lutetium(¹⁷⁷Lu). Methods of administering multiple doses of the antibodymolecules of the invention coupled to ¹⁷⁷Lu and methods of evaluatingmultiple dose regimens are described herein.

Other features and advantages of the instant invention will become moreapparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depict the amino acid sequence of murine J591 heavy andlight chain variable region, respectively. The location of the CDRs isindicated in the Figures; the amino acid numbering is according theKabat numbering (see, Kabat, E. A., et al. (1991) Sequences of Proteinsof Immunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NM Publication No. 91-3242). Note that the CDRs areconsidered to encompass the Chothia loops and the Kabat hypervariableregions together and the sequences have been annotated accordingly.Heavy Chain: CDR1 is depicted in SEQ ID NO:1; CDR2 is depicted in SEQ IDNO:2; CDR3 is depicted in SEQ ID NO:3; the framework excluding CDRregions is depicted in SEQ ID NO:7; and the framework including CDRregions is depicted in SEQ ID NO:19. Light Chain: CDR1 is depicted inSEQ ID NO:4; CDR2 is depicted in SEQ ID NO:5; CDR3 is depicted in SEQ IDNO:6; the framework excluding CDR regions is depicted in SEQ ID NO:8;and the framework including CDR regions is depicted in SEQ ID NO:20.

FIGS. 2A-2B depict the amino acid sequence of the deimmunized J591 heavyand light chain variable region, respectively. The location of the CDRsis indicated in the Figures; the amino acid numbering is according theKabat numbering (see, Kabat, E. A., et al. (1991) supra). Note that theCDRs are considered to encompass the Chothia loops and the Kabathypervariable regions together and the sequences have been annotatedaccordingly. Heavy Chain: CDR1 is depicted in SEQ ID NO:1; CDR2 isdepicted in SEQ ID NO:2; CDR3 is depicted in SEQ ID NO:3; framework 1 isdepicted in SEQ ID NO:9; framework 2 is depicted in SEQ ID NO:10;framework 3 is depicted in SEQ ID NO:11; framework 4 is depicted in SEQID NO:12; the framework excluding CDR regions is depicted in SEQ IDNO:17; and the framework including CDR regions is depicted in SEQ IDNO:21. Light Chain: CDR1 is depicted in SEQ ID NO:4; CDR2 is depicted inSEQ ID NO:5; CDR3 is depicted in SEQ ID NO:6; framework 1 is depicted inSEQ ID NO:13; framework 2 is depicted in SEQ ID NO:14; framework 3 isdepicted in SEQ ID NO:15; framework 4 is depicted in SEQ ID NO:16; theframework excluding CDR regions is depicted in SEQ ID NO:18; and theframework including CDR regions is depicted in SEQ ID NO:22.

FIGS. 3A-3B depict an alignment of the murine J591 and deimmunized heavychain variable regions (3A; SEQ ID NO:19 and 21, respectively) and lightchain variable regions (3B; SEQ ID NO:20 and 22, respectively).Potential T cell epitopes (identified using a peptide threading program)in murine J591 VH and VK are shown in FIGS. 3A-3B, respectively. The13-mer peptides predicted to bind to MHC class II are indicated by theunderline; the CDRs are located at residues 26 to 35, 50 to 66, and99-104 of FIG. 3A and residues 24 to 34, 50 to 56, and 89 to 97 of FIG.3B; and residues altered in the deimmunized heavy and light chainvariable regions are boxed. Where possible, amino acid substitutions arethose commonly used in human germline VH regions. The amino acidnumbering is linear, not according to Kabat.

FIGS. 4A-4B depict the nucleotide sequences of the deimmunized J591heavy and light chain variable region, respectively. FIG. 4A shows analignment of the coding and noncoding nucleotide strands of deimmunizedJ591 heavy chain variable region (SEQ ID NOs:23 and 24, respectively)with the corresponding amino acid sequence (SEQ ID NO:27). FIG. 4B showsan alignment of the coding and noncoding nucleotide strands ofdeimmunized J591 light chain variable region (SEQ ID NOs:25 and 26,respectively) with the corresponding amino acid sequence (SEQ ID NO:28).The location of the signal peptide and CDRs 1-3 is indicated in eachalignment.

FIG. 5 depicts an alignment of the amino acid sequences for the murineand several deimmunized heavy chain variable regions of the J415antibody. The murine amino acid sequence is shown as J415VH (SEQ IDNO:47); the deimmunized sequences are depicted as J415DIVH1 (amino acidresidues 18 to 133 of SEQ ID NO:54), J415DIVH2 (SEQ ID NO:59), J415DIVH3(SEQ ID NO:60), and J415DIVH4 (SEQ ID NO:49). The preferred sequence isJ415DIVH4 (SEQ ID NO:49). The amino acid replacements are indicated bythe boxed residues. A consensus sequence is labeled “majority” (SEQ IDNO:61).

FIG. 6 depicts an alignment of the amino acid sequences for the murineand several deimmunized light chain variable regions of the J415antibody. The murine amino acid sequence is shown as J415VK (SEQ IDNO:48); the deimmunized sequences are depicted as J415DIVK1 (amino acidresidues 18 to 124 of SEQ ID NO:57), J415DIVK2 (SEQ ID NO:62), J415DIVK3(SEQ ID NO:63), J415DIVK4 (SEQ ID NO:64), J415DIVK5 (SEQ ID NO:50),J415DIVK6 (SEQ ID NO:65), J415DIVK7 (SEQ ID NO:66), and J415DIVK8 (SEQID NO:67). The preferred sequence is J415DIVK5 (SEQ ID NO:50). The aminoacid replacements are indicated by the boxed residues. A consensussequence is labeled “majority” (SEQ ID NO:68).

FIG. 7A depicts the nucleic acid coding sequence, the amino acidsequence, and the nucleic acid reverse complement sequence of thedeimmunized J415 heavy chain variable region (J415DIVH1) (SEQ IDNO:53-55, respectively). The relative location of the signal sequence,intron and J415DIVH1 amino acid sequence is indicated, as well as somerestriction sites.

FIG. 7B depicts the nucleic acid coding sequence, the amino acidsequence, and the nucleic acid reverse complement sequence of the murineJ415 heavy chain variable region (SEQ ID NO:125, 47, and 126,respectively). The relative locations of the CDRs and some restrictionsites are indicated.

FIG. 7C depicts an alignment of the amino acid sequence of the murineJ415 heavy chain variable region (SEQ ID NO:47) and a consensus sequencefor Kabat subgroup murine VHIIIC (MUVHIII, SEQ ID NO:69). A consensusmajority sequence based on the alignment is also shown (SEQ ID NO:70).

FIG. 8A depicts the nucleic acid coding sequence, the amino acidsequence, and the nucleic acid reverse complement sequence of thedeimmunized J415 light chain variable region (J415DIVK1) (SEQ IDNO:56-58, respectively). The relative location of the signal sequence,intron and J415DIVK1 amino acid sequence is indicated, as well as somerestriction sites.

FIG. 8B depicts the nucleic acid coding sequence, the amino acidsequence, and the nucleic acid reverse complement sequence of the murineJ415 light chain variable region (SEQ ID NOs:127, 48, and 128,respectively). The relative locations of the CDRs and some restrictionsites are also indicated.

FIG. 8C depicts an alignment of the amino acid sequence of the murineJ415 light chain variable region (SEQ ID NO:48) and a consensus sequencefor Kabat subgroup murine variable light chain (MuVKI, SEQ ID NO:71). Aconsensus majority sequence based on the alignment is also shown (SEQ IDNO:72).

FIG. 9A depicts the nucleic acid coding sequence, the amino acidsequence, and the nucleic acid reverse complement sequence of the murineJ533 heavy chain variable region (SEQ ID NO:73-75, respectively). Therelative locations of the CDRs and restriction sites are indicated.

FIG. 9B depicts an alignment of the amino acid sequence of the murineJ533 heavy chain variable region (SEQ ID NO:74) and a consensus sequencefor Kabat subgroup murine variable heavy chain (MuVHIIA, SEQ ID NO:79).A consensus majority sequence based upon the alignment is also shown(SEQ ID NO:80).

FIG. 10A depicts the nucleic acid coding sequence, the amino acidsequence, and the nucleic acid reverse complement sequence of the murineJ533 light chain variable region (SEQ ID NO:76-78, respectively). Therelative locations of the CDRs and some restriction sites are indicated.

FIG. 10B depicts an alignment of the amino acid sequence of the murineJ533 light chain variable region (SEQ ID NO:77) and a consensus sequencefor Kabat subgroup murine MuVKIII, SEQ ID NO:81). A consensus majoritysequence based upon the alignment is also shown (SEQ ID NO:82).

FIG. 11A depicts the nucleic acid coding sequence, the amino acidsequence, and the nucleic acid reverse complement sequence of the murineE99 heavy chain variable region (SEQ ID NO:83-85, respectively). Therelative locations of the CDRs and some restriction sites are indicated.

FIG. 11B depicts an alignment of the amino acid sequence of the murineE99 heavy chain variable region (SEQ ID NO:84) and a consensus sequencefor Kabat subgroup murine variable heavy chain (MuVHIB, SEQ ID NO:89). Aconsensus majority sequence based upon the alignment is also shown (SEQID NO:90).

FIG. 12A depicts the nucleic acid coding sequence, the amino acidsequence, and the nucleic acid reverse complement sequence of the murineE99 light chain variable region (SEQ ID NO:86-88, respectively). Therelative locations of the CDRs and some restriction sites are indicated.

FIG. 12B depicts an alignment of the amino acid sequence of the murineE99 light chain variable region (SEQ ID NO:87) and a consensus sequencefor Kabat subgroup murine variable light chain (MuVKI, SEQ ID NO:91). Aconsensus majority sequence based upon the alignment is also shown (SEQID NO:92).

FIGS. 13A and B depict serum PSA levels as a function of time for twopatients that were treated with a single dose of ⁹⁰Y-DOTA-deJ591. Day 0corresponds to the day on which the ⁹⁰Y-DOTA-deJ591 was administered.

FIG. 14 depicts the serum PSA levels as a function of time for a patientthat was treated with a single dose of ¹⁷⁷Lu-DOTA-deJ591. Day 0corresponds to the day on which the ¹⁷⁷Lu-DOTA-deJ591 was administered.

FIG. 15 depicts the chemical structures of DM1 and maytansine, a relatedmolecule that lacks the thiol reactive group of DM1 used to conjugateDM1 to antibodies.

FIGS. 16A and B depict CWR22 xenograft growth in C.B-17 Scid Mice. 16A.depicts mean tumor volume (mm³) after the administration every day forfive cycles of unconjugated DM1 or deJ591-DM1 at a dose of 240 μg/kg DM1equivalents (eq.), or dcJ591-DM1 at a dose of 120m/kg DM1 eq. A controlvehicle was also administered. 16B depicts depicts mean tumor volume(mm³) after the administration every three days for five cycles ofunconjugated DM1, or deJ591-DM1 at a dose of 240 μg/kg DM1 eq., ordeJ591-DM1 at a dose of 120 μg/kg DM1 eq.

FIG. 17 depicts the effect of deJ591-DM1 on serum PSA concentrations andmean tumor volume (mm³) in Scid mice with PSMA-Positive CWR22xenografts. DeJ591-DM1 was administered every three days for five cyclesof 240 μg/kg DM1-equivalents. The first course began on day 0, and thesecond course began on day 52. Bars show serum PSA concentrations; thearrow shows the start of the second course.

FIG. 18 depicts CWR22 xenograft growth in C.B-17 Scid mice receivingdeJ591-DM1 at a dosage of 12.93 mg/kg deJ591-DM1 (240 μg/kgDM1-equivalents) at different dosing schedules of 7, 14, 21, or 28 daysfor five cycles.

FIG. 19 is a graph of MALDI-TOF MS data for naked deJ591 and conjugateddeJ591 samples for 2+ charge state peaks. The data was baselinecorrected and processed using noise-filter smooth (factor 0.9); (a)naked deJ591 (left trace), (b) conjugation of DOTA-deJ591 batch 108A(center trace), mass difference from naked deJ591 shows 4.8 DOTA foreach deJ591, (c) conjugation of DOTA-deJ591 batch 110A (right trace).The mass difference from naked deJ591 shows 8.7 DOTA for each deJ591.

FIG. 20A illustrates real time kinetics of DOTA-NHS (Lot B280A)hydrolysis in water.

FIG. 20B is a plot of concentration of DOTA-NHS vs. time, indicating therate of hydrolysis in water.

FIG. 21 is a graph of a MALDI MS spectra of naked deJ591 and two batchesof conjugations of DOTA-J591 after controlling the input ratio ofDOTA-NHS to deJ591 at 20:1. (a) naked deJ591 (Red Trace); (b)conjugation batch 102802a, 5.7 DOTA for each deJ591 (Green Trace); (c)conjugation batch 102802b, 6.0 DOTA for each deJ591 (Blue trace).

FIG. 22 is a series of line graphs showing the level of DOTA conjugationratios; top trace shows naked deJ591, middle trace shows Gaussiandeconvolution for DOTA conjugated deJ591, bottom trace displays peakfitting for middle trace.

FIG. 23 is a line graph of a comparison of naked deJ591 control peakwith overlay of DOTA conjugated deJ591, displaying Gaussian deconvolutedpeaks indicating levels of DOTA incorporation.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides, inter alia, antibodies, e.g., modifiedantibodies, or antigen-binding fragments thereof, to the extracellulardomain of human prostate specific membrane antigen (PSMA). The modifiedanti-PSMA antibodies, or antigen-binding fragments thereof, have beenrendered less immunogenic compared to their unmodified counterparts to agiven species, e.g., a human. Human PSMA is expressed on the surface ofnormal, benign hyperplastic epithelial cells (e.g., benign prostatesecretory-acinar epithelium), and cancerous prostate epithelial cells(e.g., prostatic intraepithelial neoplasia and prostaticadenocarcinoma), as well as vascular endothelial cells proximate tocancerous cells, e.g., renal, urothelial (e.g., bladder), testicular,colon, rectal, lung (e.g., non-small cell lung carcinoma), breast,liver, neural (e.g., neuroendocrine), glial (e.g., glioblastoma),pancreatic (e.g., pancreatic duct), melanoma (e.g., malignant melanoma),or soft tissue sarcoma cancerous cells. The antibodies, e.g., themodified antibodies, of the invention bind to the cell surface of cellsthat express PSMA. PSMA is normally recycled from the cell membrane intothe cell. Thus, the antibodies of the invention are internalized withPSMA through the process of PSMA recirculation, thereby permittingdelivery of an agent conjugated to the antibody, e.g., a labeling agent,a cytotoxic agent, or a viral particle (e.g., a viral particlecontaining genes that encode cytotoxic agents, e.g., apoptosis-promotingfactors).

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, “PSMA” or “prostate-specific membrane antigen” proteinrefers to mammalian PSMA, preferably human PSMA protein. Human PSMAincludes the two protein products, PSMA and PSM′, encoded by the twoalternatively spliced mRNA variants (containing about 2,653 and 2,387nucleotides, respectively) of the PSMA cDNA disclosed in Israeli et al.(1993) Cancer Res. 53:227-230; Su et al. (1995) Cancer Res.55:1441-1443; U.S. Pat. No. 5,538,866, U.S. Pat. No. 5,935,818, and WO97/35616, the contents of which are hereby incorporated by reference.The long transcript of PSMA encodes a protein product of about 100-120kDa molecular weight characterized as a type II transmembrane receptorhaving sequence homology with the transferrin receptor and havingNAALADase activity (Carter et al. (1996) Proc. Natl. Acad. Sci. USA93:749-753). Accordingly, the term “human PSMA” refers to at least twoprotein products, human PSMA and PSM′, which have or are homologous to(e.g., at least about 85%, 90%, 95% identical to) an amino acid sequenceas shown in Israeli et al. (1993) Cancer Res. 53:227-230; Su et al.(1995) Cancer Res. 55:1441-1443; U.S. Pat. No. 5,538,866, U.S. Pat. No.5,935,818, and WO97/35616; or which is encoded by (a) a naturallyoccurring human PSMA nucleic acid sequence (e.g., Israeli et al. (1993)Cancer Res. 53:227-230 or U.S. Pat. No. 5,538,866); (b) a nucleic acidsequence degenerate to a naturally occurring human PSMA sequence; (c) anucleic acid sequence homologous to (e.g., at least about 85%, 90%, 95%identical to) the naturally occurring human PSMA nucleic acid sequence;or (d) a nucleic acid sequence that hybridizes to one of the foregoingnucleic acid sequences under stringent conditions, e.g., highlystringent conditions.

An “anti-PSMA antibody” is an antibody that interacts with (e.g., bindsto) PSMA, preferably human PSMA protein. Preferably, the anti-PSMAantibody interacts with, e.g., binds to, the extracellular domain ofPSMA, e.g., the extracellular domain of human PSMA located at aboutamino acids 44-750 of human PSMA (amino acid residues correspond to thehuman PSMA sequence disclosed in U.S. Pat. No. 5,538,866). In oneembodiment, the anti-PSMA antibody binds all or part of the epitope ofan antibody described herein, e.g., J591, E99, J415, and J533. Theanti-PSMA antibody can inhibit, e.g., competitively inhibit, the bindingof an antibody described herein, e.g., J591, E99, J415, and J533, tohuman PSMA. An anti-PSMA antibody may bind to an epitope, e.g., aconformational or a linear epitope, which epitope when bound preventsbinding of an antibody described herein, J591, E99, J415, and J533. Theepitope can be in close proximity spatially or functionally-associated,e.g., an overlapping or adjacent epitope in linear sequence orconformationally to the one recognized by the J591, E99, J415, or J533antibody. In one embodiment, the anti-PSMA antibody binds to an epitopelocated wholly or partially within the region of about amino acids 120to 500, preferably 130 to 450, more preferably, 134 to 437, or 153 to347, of human PSMA (amino acid residues correspond to the human PSMAsequence disclosed in U.S. Pat. No. 5,538,866). Preferably, the epitopeincludes at least one glycosylation site, e.g., at least one N-linkedglycosylation site (e.g., an asparagine residue located at about aminoacids 190-200, preferably at about amino acid 195, of human PSMA; aminoacid residues correspond to the human PSMA sequence disclosed in U.S.Pat. No. 5,538,866).

Cell lines that produce anti-PSMA antibodies, e.g., murine and modifiedanti-PSMA antibodies, described herein have been deposited with theATCC. The ATCC designations of the cell lines that produce each of theanti-PSMA antibodies are listed in Table 7.

TABLE 7 Anti-PSMA Antibody ATCC Designation E99 HB-12101 J415 HB-12109J533 HB-12127 J591 HB-12126 deJ591 PTA-3709 deJ415 PTA-4174

In a preferred embodiment, the interaction, e.g., binding, occurs withhigh affinity (e.g., affinity constant of at least 10⁷ M⁻¹, preferably,between 10⁸ M⁻¹ and 10¹⁰, or about 10⁹ M⁻¹) and specificity. Preferably,the anti-PSMA antibody treats, e.g., ablates or kills, a cell, e.g., aPSMA-expressing cell (e.g., a prostatic or cancerous cell). Themechanism by which the anti-PSMA antibody treats, e.g., ablates orkills, the cell is not critical to the practice of the invention. In oneembodiment, the anti-PSMA antibody may bind to and be internalized withthe PSMA expressed in the cells and/or vascular endothelial cellsproximate to the cells. In those embodiments, the anti-PSMA antibody canbe used to target a second moiety, e.g., a labeling agent, a labelingagent, or a viral agent, to the cell. In other embodiments, theanti-PSMA antibody may mediate host-mediated-killing, e.g., complement-or ADCC-mediated killing, of the cell and/or the vascular cell proximatethereto, upon binding to the extracellular domain of PSMA. The cell canbe killed directly by the anti-PSMA antibody by binding directly to thecell or the vascular endothelial cells proximate thereto. Alternatively,the anti-PSMA antibody can treat, e.g., kill or ablate, or otherwisechange the properties of the vascular endothelial cells to which itbinds so that blood flow to the cells proximate thereto is reduced,thereby causing the cells to be killed or ablated. Examples of anti-PSMAantibodies include, e.g., monospecific, monoclonal (e.g., human),recombinant or modified, e.g., chimeric, CDR-grafted, humanized,deimmunized, and in vitro generated anti-PSMA antibodies.

As used herein, the term “treat” or “treatment” is defined as theapplication or administration of an anti-PSMA antibody or antigenbinding fragment thereof to a subject, e.g., a patient, or applicationor administration to an isolated tissue or cell from a subject, e.g., apatient, which is returned to the patient. The anti-PSMA antibody orantigen binding fragment thereof, can be administered alone or incombination with, a second agent. The subject can be a patient having adisorder (e.g., a disorder as described herein), a symptom of a disorderor a predisposition toward a disorder. The treatment can be to cure,heal, alleviate, relieve, alter, remedy, ameliorate, palliate, improveor affect the disorder, the symptoms of the disorder or thepredisposition toward the disorder. While not wishing to be bound bytheory treating is believed to cause the inhibition, ablation, orkilling of a cell in vitro or in vivo, or otherwise reducing capacity ofa cell, e.g., an aberrant cell, to mediate a disorder, e.g., a disorderas described herein (e.g., a cancer or prostatic disorder).

As used herein, an amount of an anti-PSMA antibody effective to treat adisorder, or a “therapeutically effective amount” refers to an amount ofthe antibody which is effective, upon single or multiple doseadministration to a subject, in treating a cell, e.g., a prostatic orcancer cell (e.g., a PSMA-expressing prostatic or cancer cell, or avascular cell proximate thereto), or in prolonging curing, alleviating,relieving or improving a subject with a disorder as described hereinbeyond that expected in the absence of such treatment. As used herein,“inhibiting the growth” of the neoplasm refers to slowing, interrupting,arresting or stopping its growth and metastases and does not necessarilyindicate a total elimination of the neoplastic growth.

As used herein, an amount of an anti-PSMA antibody effective to preventa disorder, or a “a prophylactically effective amount” of the antibodyrefers to an amount of an anti-PSMA antibody, e.g., an anti-PSMAantibody as described herein, which is effective, upon single- ormultiple-dose administration to the subject, in preventing or delayingthe occurrence of the onset or recurrence of a disorder, e.g., a canceror prostatic disorder as described herein, or treating a symptomthereof.

The terms “induce”, “inhibit”, “potentiate”, “elevate”, “increase”,“decrease” or the like, e.g., which denote quantitative differencesbetween two states, refer to a difference, e.g., a statistically orclinically significant difference, between the two states. For example,“an amount effective to inhibit the proliferation of the PSMA-expressinghyperproliferative cells” means that the rate of growth of the cellswill be different, e.g., statistically different, from the untreatedcells.

As used herein, “specific binding” refers to the property of theantibody to: (1) to bind to PSMA, e.g., human PSMA protein, with anaffinity of at least 1×10⁷ M⁻¹, and (2) preferentially bind to PSMA,e.g., human PSMA protein, with an affinity that is at least two-fold,50-fold, 100-fold, 1000-fold, or more greater than its affinity forbinding to a non-specific antigen (e.g., BSA, casein) other than PSMA.

As used herein, the term “antibody” refers to a protein comprising atleast one, and preferably two, heavy (H) chain variable regions(abbreviated herein as VH), and at least one and preferably two light(L) chain variable regions (abbreviated herein as VL). The VH and VLregions can be further subdivided into regions of hypervariability,termed “complementarity determining regions” (“CDR”), interspersed withregions that are more conserved, termed “framework regions” (FR). Theextent of the framework region and CDRs has been precisely defined (see,Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol.196:901-917, which are incorporated herein by reference). Preferably,each VH and VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4.

The VH or VL chain of the antibody can further include all or part of aheavy or light chain constant region. In one embodiment, the antibody isa tetramer of two heavy immunoglobulin chains and two lightimmunoglobulin chains, wherein the heavy and light immunoglobulin chainsare inter-connected by, e.g., disulfide bonds. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. The light chainconstant region is comprised of one domain, CL. The variable region ofthe heavy and light chains contains a binding domain that interacts withan antigen. The constant regions of the antibodies typically mediate thebinding of the antibody to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system. The term “antibody”includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (aswell as subtypes thereof), wherein the light chains of theimmunoglobulin may be of types kappa or lambda.

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. The recognized human immunoglobulin genes include the kappa,lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Full-length immunoglobulin “lightchains” (about 25 Kd or 214 amino acids) are encoded by a variableregion gene at the NH2-terminus (about 110 amino acids) and a kappa orlambda constant region gene at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (about 50 Kd or 446 amino acids), aresimilarly encoded by a variable region gene (about 116 amino acids) andone of the other aforementioned constant region genes, e.g., gamma(encoding about 330 amino acids). The term “immunoglobulin” includes animmunoglobulin having: CDRs from a non-human source, e.g., from anon-human antibody, e.g., from a mouse immunoglobulin or anothernon-human immunoglobulin, from a consensus sequence, or from a sequencegenerated by phage display, or any other method of generating diversity;and having a framework that is less antigenic in a human than anon-human framework, e.g., in the case of CDRs from a non-humanimmunoglobulin, less antigenic than the non-human framework from whichthe non-human CDRs were taken. The framework of the immunoglobulin canbe human, humanized non-human, e.g., a mouse, framework modified todecrease antigenicity in humans, or a synthetic framework, e.g., aconsensus sequence. These are sometimes referred to herein as modifiedimmunoglobulins. A modified antibody, or antigen binding fragmentthereof, includes at least one, two, three or four modifiedimmunoglobulin chains, e.g., at least one or two modified immunoglobulinlight and/or at least one or two modified heavy chains. In oneembodiment, the modified antibody is a tetramer of two modified heavyimmunoglobulin chains and two modified light immunoglobulin chains.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

The term “antigen-binding fragment” of an antibody (or simply “antibodyportion,” or “fragment”), as used herein, refers to a portion of anantibody which specifically binds to PSMA (e.g., human PSMA), e.g., amolecule in which one or more immunoglobulin chains is not full lengthbut which specifically binds to PSMA (e.g., human PSMA protein).Examples of binding fragments encompassed within the term“antigen-binding fragment” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR) havingsufficient framework to specifically bind, e.g., an antigen bindingportion of a variable region. An antigen binding portion of a lightchain variable region and an antigen binding portion of a heavy chainvariable region, e.g., the two domains of the Fv fragment, VL and VH,can be joined, using recombinant methods, by a synthetic linker thatenables them to be made as a single protein chain in which the VL and VHregions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding fragment” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

The term “monospecific antibody” refers to an antibody that displays asingle binding specificity and affinity for a particular target, e.g.,epitope. This term includes a “monoclonal antibody” or “monoclonalantibody composition,” which as used herein refer to a preparation ofantibodies or fragments thereof of single molecular composition.

The term “recombinant” antibody, as used herein, refers to antibodiesthat are prepared, expressed, created or isolated by recombinant means,such as antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial antibody library, antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant antibodies include humanized, CDRgrafted, chimeric, deimmunized, in vitro generated (e.g., by phagedisplay) antibodies, and may optionally include constant regions derivedfrom human germline immunoglobulin sequences.

As used herein, the term “substantially identical” (or “substantiallyhomologous”) is used herein to refer to a first amino acid or nucleotidesequence that contains a sufficient number of identical or equivalent(e.g., with a similar side chain, e.g., conserved amino acidsubstitutions) amino acid residues or nucleotides to a second amino acidor nucleotide sequence such that the first and second amino acid ornucleotide sequences have similar activities. In the case of antibodies,the second antibody has the same specificity and has at least 50% of theaffinity of the same.

Calculations of “homology” between two sequences cab be performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent homology between twoamino acid sequences is determined using the Needleman and Wunsch(1970), J. Mol. Biol. 48:444-453, algorithm which has been incorporatedinto the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent homology between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularlypreferred set of parameters (and the one that should be used if thepractitioner is uncertain about what parameters should be applied todetermine if a molecule is within a homology limitation of theinvention) are a Blossum 62 scoring matrix with a gap penalty of 12, agap extend penalty of 4, and a frameshift gap penalty of 5.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. Specific hybridizationconditions referred to herein are as follows: 1) low stringencyhybridization conditions in 6× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); 2) medium stringency hybridization conditions in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC atabout 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C.; and preferably 4) very high stringency hybridization conditions are0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washesat 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are thepreferred conditions and the ones that should be used unless otherwisespecified.

It is understood that the antibodies and antigen binding fragmentthereof of the invention may have additional conservative ornon-essential amino acid substitutions, which do not have a substantialeffect on the polypeptide functions. Whether or not a particularsubstitution will be tolerated, i.e., will not adversely affect desiredbiological properties, such as binding activity can be determined asdescribed in Bowie, J U et al. (1990) Science 247:1306-1310. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of the binding agent, e.g., the antibody,without abolishing or more preferably, without substantially altering abiological activity, whereas an “essential” amino acid residue resultsin such a change.

Anti-PSMA Antibodies

Many types of anti-PSMA antibodies, or antigen-binding fragmentsthereof, are useful in the methods of this invention. The antibodies canbe of the various isotypes, including: IgG (e.g., IgG1, IgG2, IgG3,IgG4), IgM, IgA1, IgA2, IgD, or IgE. Preferably, the antibody is an IgGisotype, e.g., IgG1. The antibody molecules can be full-length (e.g., anIgG1 or IgG4 antibody) or can include only an antigen-binding fragment(e.g., a Fab, F(ab′)₂, Fv or a single chain Fv fragment). These includemonoclonal antibodies, recombinant antibodies, chimeric antibodies,humanized antibodies, dimmunized antibodies, and human antibodies, aswell as antigen-binding fragments of the foregoing.

Monoclonal anti-PSMA antibodies can be used in the methods of theinvention. Preferably, the monoclonal antibodies bind to theextracellular domain of PSMA (i.e., an epitope of PSMA located outsideof a cell). Examples of preferred murine monoclonal antibodies to humanPSMA include, but are not limited to, E99, J415, J533 and J591, whichare produced by hybridoma cell lines having an ATCC Accession NumberHB-12101, HB-12109, HB-12127, and HB-12126, respectively, all of whichare disclosed in U.S. Pat. No. 6,107,090 and U.S. Pat. No. 6,136,311,the contents of which are expressly incorporated by reference. Mostpreferably, the murine monoclonal antibody is J591, produced byHB-12126.

Additional monoclonal antibodies to PSMA can be generated usingtechniques known in the art. Monoclonal antibodies can be produced by avariety of techniques, including conventional monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein, Nature 256:495 (1975). See generally, Harlow, E.and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

Useful immunogens for the purpose of this invention include PSMA (e.g.,human PSMA)-bearing cells (e.g., a prostate tumor cell line, e.g., LNCapcells, or fresh or frozen prostate tumor cells); membrane fractions ofPSMA-expressing cells (e.g., a prostate tumor cell line, e.g., LNCapcells, or fresh or frozen prostate tumor cells); isolated or purifiedPSMA, e.g., human PSMA protein (e.g., biochemically isolated PSMA, or aportion thereof, e.g., the extracellular domain of PSMA). Techniques forgenerating antibodies to PSMA are described in U.S. Pat. No. 6,107,090,U.S. Pat. No. 6,136,311, the contents of all of which are expresslyincorporated by reference.

Human monoclonal antibodies (mAbs) directed against human proteins canbe generated using transgenic mice carrying the human immunoglobulingenes rather than the mouse system. Splenocytes from these transgenicmice immunized with the antigen of interest are used to producehybridomas that secrete human mAbs with specific affinities for epitopesfrom a human protein (see, e.g., Wood et al. International ApplicationWO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg etal. International Application WO 92/03918; Kay et al. InternationalApplication 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green,L. L. et al. 1994 Nature Genet. 7:13-21; Morrison, S. L. et al. 1994Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 YearImmunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman etal. 1991 Eur J Immunol 21:1323-1326).

Anti-PSMA antibodies or fragments thereof useful in the presentinvention may also be recombinant antibodies produced by host cellstransformed with DNA encoding immunoglobulin light and heavy chains of adesired antibody. Recombinant antibodies may be produced by knowngenetic engineering techniques. For example, recombinant antibodies maybe produced by cloning a nucleotide sequence, e.g., a cDNA or genomicDNA, encoding the immunoglobulin light and heavy chains of the desiredantibody. The nucleotide sequence encoding those polypeptides is theninserted into expression vectors so that both genes are operativelylinked to their own transcriptional and translational expression controlsequences. The expression vector and expression control sequences arechosen to be compatible with the expression host cell used. Typically,both genes are inserted into the same expression vector. Prokaryotic oreukaryotic host cells may be used.

Expression in eukaryotic host cells is preferred because such cells aremore likely than prokaryotic cells to assemble and secrete a properlyfolded and immunologically active antibody. However, any antibodyproduced that is inactive due to improper folding may be renaturableaccording to well known methods (Kim and Baldwin, “SpecificIntermediates in the Folding Reactions of Small Proteins and theMechanism of Protein Folding”, Ann. Rev. Biochem. 51, pp. 459-89(1982)). It is possible that the host cells will produce portions ofintact antibodies, such as light chain dimers or heavy chain dimers,which also are antibody homologs according to the present invention.

It will be understood that variations on the above procedure are usefulin the present invention. For example, it may be desired to transform ahost cell with DNA encoding either the light chain or the heavy chain(but not both) of an antibody. Recombinant DNA technology may also beused to remove some or all of the DNA encoding either or both of thelight and heavy chains that is not necessary for PSMA binding, e.g., theconstant region may be modified by, for example, deleting specific aminoacids. The molecules expressed from such truncated DNA molecules areuseful in the methods of this invention. In addition, bifunctionalantibodies may be produced in which one heavy and one light chain areanti-PSMA antibody and the other heavy and light chain are specific foran antigen other than PSMA, or another epitope of PSMA.

Chimeric antibodies can be produced by recombinant DNA techniques knownin the art. For example, a gene encoding the Fc constant region of amurine (or other species) monoclonal antibody molecule is digested withrestriction enzymes to remove the region encoding the murine Fc, and theequivalent portion of a gene encoding a human Fc constant region issubstituted (see Robinson et al., International Patent PublicationPCT/US86/02269; Akira, et al., European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., InternationalApplication WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabillyet al., European Patent Application 125,023; Better et al. (1988 Science240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987,J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimuraet al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature314:446-449; and Shaw et al., 1988, Natl Cancer Inst. 80:1553-1559).

An antibody or an immunoglobulin chain can be humanized by methods knownin the art. Once the murine antibodies are obtained, the variableregions can be sequenced. The location of the CDRs and frameworkresidues can be determined (see, Kabat, E. A., et al. (1991) Sequencesof Proteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated hereinby reference). The light and heavy chain variable regions can,optionally, be ligated to corresponding constant regions.

Murine anti-PSMA antibodies can be sequenced using art-recognizedtechniques. As an example, hybridomas expressing murine hybridomas J533,J415 and E99 were propagated in culture in RPMI 1640 medium supplementedwith 10% fetal calf serum. The isotype of the antibodies secreted wasconfirmed as IgG1*, IgG1*, and IgG3* respectively. These monoclonalantibodies, like J591, bind to the external domain of prostate specificmembrane antigen. J591, J533 and E99 recognize the same epitope, whileJ415 recognizes an independent epitope. Total RNA for each monoclonalwas prepared from 10⁷ hybridoma cells. V_(H) and V_(K) cDNA was preparedusing reverse transcriptase and mouse constant region and mouse IgGconstant region primers. The first strand cDNAs were amplified by PCRusing a variety of mouse signal sequence primers (6 for V_(H) and 7 forV_(K)). The amplified DNAs were gel-purified and cloned into the vectorpT7Blue. The V_(H) and V_(K) clones obtained were screened for correctinserts by PCR and the DNA sequence of selected clones determined by thedideoxy chain termination method (see Table 7).

The DNA and amino acid sequences for the heavy and light chain variableregions from J415 were obtained and are shown in FIGS. 7B (V_(H)) and 8B(V_(K)) (also, see Table 5). The location of the CDRs is shown. J415V_(H) can be assigned to Mouse Heavy Chains Subgroup IIIC (Kabat E A etal; ibid). The sequence of J415 V_(H) compared to the consensus sequencefor this subgroup is shown in FIG. 7C. J415 V_(K) can be assigned toMouse Kappa Chains Subgroup I (Kabat E A et al; ibid). The sequence ofJ415 V_(K) compared to the consensus sequence for this subgroup is shownin FIG. 8C.

The DNA and amino acid sequences encoding the heavy and light chainvariable regions J533 were obtained and are shown in FIGS. 9A (V_(H))and 10A (V_(K)) (see also Table 5). The location of the CDRs is shown ineach figure. J533 V_(H) can be assigned to Mouse Heavy Chains SubgroupIIA (Kabat E A et al; Sequences of proteins of Immunological Interest,US Department of Health and Human Services, 1991). The sequence of J533V_(H) compared to the consensus sequence for this subgroup is shown inFIG. 9B. J533 V_(K) can be assigned to Mouse Kappa Chains Subgroup III(Kabat E A et al; ibid). The sequence of J533 V_(K) compared to theconsensus sequence for this subgroup is shown in FIG. 10B.

The DNA and amino acid sequences of the heavy and light chain variableregions of E99 were obtained and are shown in FIGS. 11A (V_(H)) and 12A(V_(K)) (sec also Table 5). The location of the CDRs is shown. E99 V_(H)can be assigned to Mouse Heavy Chains Subgroup IB (Kabat E A et al;ibid). The sequence of E99 V_(H) compared to the consensus sequence forthis subgroup is shown in FIG. 11B. E99 V_(K) can be assigned MouseKappa Chains Subgroup I (Kabat E A et al; ibid). The sequence of E99V_(K) compared to the consensus sequence for this subgroup is shown inFIG. 12B.

The amino acid sequence and nucleotide sequences encoding the variableregion of antibodies J415, deJ415, J591, deJ591, J533 and E99 areprovided below in Table 8.

TABLE 8 Antibody variable chain sequences SEQ ID NAME Organism FIG. NO:SEQUENCe V_(H) J415 Mus musculus FIG. 7B 125gaagtgaagcttgaggagtctggaggaggcttggtgcaacctggaggatccatgaaactctcctgtgttgcctctggattcactttcagtaattactggatgaactgggtccgccagtctccagagaaggggcttgagtgggttgctgaaattagatcgcaatctaataattttgcaacacattatgcggagtctgtgaaagggagggtcatcatctcaagagatgattccaagagtagtgtctacctgcaaatgaacaacttgagagctgaagacactggcatttattactgtaccaggcgatggaataatttctggggccaaggcaccactctcacagtctcctca V_(H) Variable  Mus musculus FIG. 1A  19EVQLQQSGPELKKPGTSVRISCKTS Region J591 GYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKS SSTAYMFLRSLTSEDSAVYYCAAG WNFDYWGQGTTLTVSSV_(H) J415 Mus musculus FIG. 7B 126tgaggagactgtgagagtggtgccttggccccagaaat (complementary tattccatcgcctggtacagtaataaatgccagtgtcttca strandgctctcaagttgttcatttgcaggtagacactactcttgga of SEQ ID NO:atcatctcttgagatgatgaccctccctttcacagactccg 125)cataatgtgttgcaaaattattagattgcgatctaatttcagcaacccactcaagccccttctctggagactggcggacccagttcatccagtaattactgaaagtgaatccagaggcaacacaggagagtttcatggatcctccaggttgcaccaag cctcctccagactcctcaagcttcacttcV_(L) J415 Mus musculus FIG. 8B 127aacattgtaatgacccaatttcccaaatccatgtccatttcagtaggagagagggtcaccttgacctgcaaggccagtgagaatgtgggtacttatgtgtcctggtatcaacagaaaccagaacagtctcctaagatgttgatatacggggcatccaaccggttcactggggtccccgatcgcttcacaggcagtggatctgcaacagatttcattctgaccatcagcagtgtgcagactgaagaccttgtagattattactgtggacagagttacacctttccgtacacgttcggaggggggaccaagctgg aaatgaag V_(L) Variable Mus musculus FIG. 1B  20 DIVMTQSHKFMSTSVGDRVSIICKA Region J591SQDVGTAVDWYQQKPGQSPKLLIY WASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFG AGTMLDLK V_(L) J415 Mus musculus FIG. 8B 128cttcatttccagcttggtcccccctccgaacgtgtacgga (complementary aaggtgtaactctgtccacagtaataatctacaaggtctt strandcagtctgcacactgctgatggtcagaatgaaatctgttgc of SEQ ID NO: agatccactgcctgtgaagcgatcggggaccccagtga 127)accggttggatgccccgtatatcaacatcttaggagactgttctggtttctgttgataccaggacacataagtacccacattctcactggccttgcaggtcaaggtgaccctctctcctactgaaatggacatggatttgggaaattgggtcattacaa tgtt V_(H) Variable Artificial- FIG. 2A  21 EVQLVQSGPEVKKPGATVKISCKTS Region (Deimm)deimmunized heavy GYTFTEYTIHWVKQAPGKGLEWIG J591 chainNINPNNGGTTYNQKFEDKATLTVD J591 KSTDTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTLLTVSS V_(L) Variable  Artificial- FIG. 2B  22DIQMTQSPSSLSTSVGDRVTLTCKA Region (Deimm) deimmunized lightSQDVGTAVDWYQQKPGPSPKLLIY J591 chain WASTRHTGIPSRFSGSGSGTDFTLTI J591SSLQPEDFADYYCQQYNSYPLTFGP GTKVDIK V_(H) Deimmunized Artificial- FIG. 4A 23 Aagcttatgaatatgcaaatcctctgaatctacatggtaa J591 deimmunized heavyatataggtttgtctataccacaaacagaaaaacatgagat CDS (122-166) & chaincacagttctctctacagttactgagcacacaggacctca CDS (249-605) J591ccatgggatggagctgtatcatcctcttcttggtagcaacagctacaggtaaggggctcacagtagcaggcttgaggtctggacatatatatgggtgacaatgacatccactttgcctttctctccacaggtgtccactccgaggtccaactggtacagtctggacctgaagtgaagaagcctggggctacagtgaagatatcctgcaagacttctggatacacattcactgaatataccatacactgggtgaagcaggcccctggaaagggccttgagtggattggaaacatcaatcctaacaatggtggtaccacctacaatcagaagttcgaggacaaggccacactaactgtagacaagtccaccgatacagcctacatggagctcagcagcctaagatctgaggatactgcagtctattattgtgcagctggaggaactagactactggggccaagggaccctgctcaccgtctcctcaggtgagtccttacaacctctctcactattcagcttaaatagatatactgcatttgagggggggaaatgtgtgtatctgaatttcaggtcatgaaggactagggacaccagggagtcagaaagggtcattgggagcccgggctgatgcagacagacatcctcagctcccagacttcat ggccagagatttataggatccV_(H) Deimmunized Artificial- FIG. 4A  24ggatcctataaatctctggccatgaagtctgggagclga (complimentary deimmunized heavy ggatgtctgtetgcatcagcccgggctcccaatgaccct strand ofchain actgactcccaaggtgtccctagtccacatgacctgaa SEQ ID NO: 23) J591attcagatacacacatttcccccccaacaaatgcagtaaa J591atctatttaagctgaatagaagagagaggagtaaggactcacctgaggagacggtgagcagggtcccttggccccagtagtcaaagttccaaccagctgcacaataatagactgcagtatcctcagatcttaggctgctgagctccatgtaggctgtatcggtggacttgtctacagttagtgtggccagtcctcgaacactgattgtaggtggtaccaccattgaaggattgatgatccaatccactcaaggccctaccaggggcctgcttcacccagtgtatggtatattcagtgaatgtgtatccagaagtcagcaggatatcacactgtagccccaggcacttcacttcaggtccagactgtaccagaggacctcggagtggacacctgtggagagaaaggcaaagtggatgtcattgtcacccatatatatgtccagacctcaagectgctaclgtgagccccttacctgtagctgagctaccaagaagaggatgatacagctccatcccatggtgaggtcctgtgtgctcagtaactgtagagagaactgtgatctcatgatactgagtggtatagacaaacctatatttaccatgtagattcagaggatttgcata ttcataagctt V_(L) DeimmunizedArtificial- FIG. 4B  25 aagcttatgaatatgcaaatcctctgaatctacatggtaaa J591deimmunized light tataggatgtctataccacaaacagaaaaacatgagatcCDS (122-166) & chain acagactctctacagttactgagcacacaggacctcacCDS (249-581) J591 catgggatggagctgtatcatcctottcaggtagcaacagctacaggtaaggggctcacagtagcaggcttgaggtctggacatatatatgggtgacaatgacatccactttgcctttctctccacaggtgtccactccgacatccagatgacccagtctccctcatccctgtccacatcagtaggagacagggtcaccctcacctgtaaggccagtcaagatgtgggtactgctgtagactggtatcaacagaaaccaggaccatctcctaaactactgatttattgggcatccactcggcacactggaatccctagtcgottctcaggcagtggatctgggacagacttcactctcaccatttctagtcttcagcctgaagactttgcagattattactgtcagcaatataacagctatcctctcacgttcggtcctgggaccaaggtggacatcaaacgtgagtagaattt aaactttgcttcctcagttggatccV_(L) Deimmunized Artificial- FIG. 4B  26ggatccaactgaggaagcaaagataaattctactcacgt (complimentary deimmunized light ttgatgtccaccaggtcccaggaccgaacgtgagagg  strand chainatagctgttatattgctgacagtaataatctgcaaagtcttc of SEQ ID NO: J591aggctgaagactagaaatggtgagagtgaagtctgtcc 25)cagatccactgcctgagaagcgactagggattccagtg J591tgccgagtggatgcccaataaatcagtagtttaggagatggtcctggtttctgttgataccagtctacagcagtacccacatcttgactggccttacaggtgagggtgaccctgtctcctactgatgtggacagggatgagggagactgggtcatctggatgtcggagtggacacctgtggagagaaaggcaaagtggatgtcattgtcacccatatatatgtccagacctcaagcctgctactgtgagccccttacctgtagctgttgctaccaagaagaggatgatacagctccatcccatggtgaggtcctgtgtgctcagtaactgtagagagaactgtgatctcatgtttttctgtttgtggtatagacaaacctatatttaccatgtagattcagaggatttgcatattcataagctt V_(H) Deimmunized Artificial- FIG. 4A  27MGWSCHLFLVATATGVHSEVQLVQ (predicted  deimmunized heavySGPEVKKPGATVKISCKTSGYTFIE a.a. of  chain YTIHWVKQAPGKGLEWIGNINPNNSEQ ID NO: 23) J591 GGTTYNQKFEDKATLTVDKSTDTA YMELSSLRSEDTAVYYCAAGWNFDJ591 YWGQGTLLTVSS V_(L) Deimmunized Artificial- FIG. 4B  28MGWSCIILFLVATATGVHSDIQMTQ (predicted  deimmunized lightSPSSLSTSVGDRVTLTCKASQDVGT a.a. of  chain AVDWYQQKPGPSPKLLIYCASTRHTSEQ ID NO: 25) J591 GIPSRFSGSGSGTDFTLTISSLQPEDF J591ADYYCQQYNSYPLTFGPGTKVDIK V_(H) Variable  Mus musculus FIG. 5  47EVKLEESGGGLVQPGGSMKLSCVA Region J415 SGFTFSNYWMNWVRQSPEKGLEWVAEIRSQSNNFATHYAESVKGRVIIS RDDSKSSVYLQMNNLRAEDTGIYY CTRRWNNFWGQGTTLTVSSV_(L) Variable  Mus musculus FIG. 6  48 NIVMTQFPKSMSISVGERVTLTCKARegion J415 SENVGTYVSWYQQKPEQSPKMLIY GASNRFTGVPDRFTGSGSATDFTETISSVQTEDEVDYYCGQSYTFPYTFGG GTKLEMK V_(H) Variable  Artificial- FIG. 5  49EVKLEESGGGLVQPGGSMKISCVAS Region (Deimm) deimmunized heavyGFTFSNYWMNWVRQSPEKGLEWV J415-4 chain AEIRSQSNNFATHYAESVKGRVIISR J415-4DDSKSSVYLQMNSLRAEDTAVYYC TRRWNNFWGQGTTVTVSS V_(L) Variable  Artificial-FIG. 6  50 NIVMTQFPKSMSASAGERMTLTCK Region (Deimm) deimmunized lightASENVGTYVSWYQQKPTQSPKMLR J415-5 chain YGASNRFTGVPDRFSGSGSGTDFILT J415-5ISSVQAEDLVDYYCGQSYTFPYTFG GGTKLEMK V_(H) Deimmunized Artificial-  51gaagtgaaacttgaggagtctggaggaggcttggtgca J415-4 deimmunized heavyacctggagggtccatgaaaalctcclgtgUgcctetgg chainattcactTtcaglaattactggatgaactgggtccgccagt J415-4ctccagagaaggggcttgagtgggttgctgaaattagatcgcaatctaataattttgcaacacattatgcggagtctgtgaaagggagggtcatcatctcaagagatgattccaagagtagtgtctacctgcaaatgaacagtttgagagctgaagacactgccgtttattactgtaccaggcgatggaataatttctggggccaaggcaccactgtcacagtctcctca V_(L) Deimmunized Artificial-  52aacattgtaatgacccaatttcccaaatccatgtccgcct J415-5 deimmunized lightcagcaggagagaggatgaccttgacctgcaaggccag chaintgagaatgtgggtacttatgtgtcctggtatcaacagaaa J415-5ccaacacagtctcctaagatgttgatatacggggcatccaaccggttcactggggtcccagatcgcttctccggcagtggatctggaacagatttcattctgaccatcagcagtgtgcaggcagaagaccttgtagattattactgtggacagagttacacctttccgtacacgttcggaggggggaccaagctgg aaatgaag V_(H) DeimmunizedArtificial- FIG. 7A  53 aagcttatgaatatgcaaatcctctgaatctacatggtaaa J415-1deimmunized heavy tataggtttgtctataccacaaacagaaaaacatgagatcCDS (122-160) & chain acagttctctctacagttactgagcacacaggacctcacCDS (249-608) J415-1 catgggatggagctgtatcatcctcttcttggtagcaacaMature (18-133) gctacaggtaaggggctcacagtagcaggcttgaggtctggacatatatatgggtgacaatgacatccactttgcctttctctccacaggtgtccactccgaagtgaaacttgaggagtctggaggaggcttggtgcaacctggagggtccatgaaaatctcctgtaaagcctctggattcactttcagtaattactggatgaactgggtccgccagactccagagaaggggcttgagtgggttgctcttattagatcgcaatctaataattttgcaacacattatgcggagtctgtgaaagggagggtcatcatctcaagagatgattccaagagtagtgtctacctgcaaatgaacagtttgagagctgaagacactgccgtttattactgtaccaggcgatggaataatttctggggccaaggcaccactgtcacagtctcctcaggtgagtccttacaacctctctcttctattcagcttaaatagattttactgcatttgttgggggggaaatgtgtgtatctgaatttcaggtcatgaaggactagggacaccttgggagtcagaaagggtcattgggagcccgggctgatgcagacagacatcctcagctcccagacttcatgg ccagagatttataggatccV_(H) Deimmunized Artificial- FIG. 7A  54 MGWSCIILFLVATGVHSEVKLEESG(predicted  deimmunized heavy GGLVQPGGSMKISCKASGFTFSNY a.a. of  chainWMNWVRQTPEKGLEWVALIRSQS SEQ ID NO: 53) J415-1 NNFATHYAESVKGRVIISRDDSKSSJ415-1 VYLQMNSLRAEDTAVYYCTRRWN NFWGQGTTVTVSS V_(H) Artificial- FIG. 7A 55 ggatcctataaatctctggccatgaagtctgggagctga Deimmunizeddeimmunized heavy ggatgtctgtctgcatcagcccgggctcccaatgaccct(complimentary  chain ttctgactcccaaggtgtccctagtccttcatgacctgaastrand of  J415-1 attcagatacacacatttcccccccaacaaatgcagtaaaSEQ ID NO: 53) atctatttaagctgaatagaagagagaggttgtaaggact J415-1cacctgaggagactgtgacagtggtgccttggccccagaaattattccatcgcctggtacagtaataaacggcagtgtcttcagctctcaaactgttcatttgcaggtagacactactcttggaatcatctcttgagatgatgaccctccattcacagactccgcataatgtgttgcaaaattattagattgcgatctaataagagcaacccactcaagccccttctctggagtctggcggacccagttcatccagtaattactgaaagtgaatccagaggctttacaggagattttcatggaccctccaggrtgcaccaagcctcctccagactcctcaagtttcacttcggagtggacacctgtggagagaaaggcaaagtggatgtcattgtcacccatatatatgtccagacctcaagcctgctactgtgagccccttacctgtagctgttgctaccaagaagaggatgatacagctccatcccatggtgaggtcctgtgtgctcagtaactgtagagagaactgtgatctcatgtttttctgtttgtggtatagacaaacctatatttaccatgtagattcagaggatttgc atattcataagctt V_(L)Artificial- FIG. 8A  56 aagcttatgaatatgcaaatcctctgaatctacatggtaaaDeimmunized deimmunized light tataggtttgtctataccacaaacagaaaaacatgagatcJ415-1 chain acagttctctctacagttactgagcacacaggacctcac CDS (122-160) &J415-1 catgggatggagctgtatcatcctcttcttggtagcaaca CDS (249-581)gctacaggtaaggggctcacagtagcaggcttgaggtctggacatatatatgggtgacaatgacatccactttgcctttctctccacaggtgtccactccaacattgtaatgacccaatcccccaaatccatgtccgcctcagcaggagagaggatgaccttgacctgcaaggccagtgagaattccggtacttatgtgtcctggtatcaacagaaaccaacacagtctcctaagatgttgatatacggggcatccaaccggttcactggggtcccagatcgcttctccggcagtggatctggaacagatttcattctgaccgccagcagtgtgcaggcagaagaccctgtagattattactgtggacagagttacacctttccgtacacgttcggaggggggaccaagctggaaatgaagcgtgagt agaatttaaactttgcttcctcagttggatccV_(L) Artificial- FIG. 8A  57 MGWSCIILFLVATGVHSNrVMTQSP Deimmunizeddeimmunized light KSMSASAGERMTLTCKASENSGTY (predicted  chainVSWYQQKPTQSPKMLIYGASNRFT a.a. of  J415-1 GVPDRFSGSGSGTDFILTASSVQAEDSEQ ID NO: 56) PVDYYCGQSYTFPYTFGGGTKLEM J415-1 K VL Artificial- FIG. 8A 58 ggatccaactgaggaagcaaagtttaaattctactcacg Deimmunizeddeimmunized light cttcatttccagcttggtcccccctccgaacgtgtacgga(complimentary  chain aaggtgtaactctgtccacagtaataatctacagggtcttstrand of  J415-1 ctgcctgcacactgctggcggtcagaatgaaatctgttc SEQ ID NO: 56)cagatccactgccggagaagcgatctgggaccccagt J415-1gaaccggttggatgccccgtatatcaacatcttaggagactgtgttggtttctgttgataccaggacacataagtaccggaattctcactggccttgcaggtcaaggtcatcctctctcctgctgaggcggacatggatttgggggattgggtcattacaatgttggagtggacacctgtggagagaaaggcaaagtggatgtcattgtcacccatatatatgtccagacctcaagcctgctactgtgagccccttacctgtagctgttgctaccaagaagaggatgatacagctccatcccatggtgaggtcctgtgtgctcagtaactgtagagagaactgtgatctcatgtttttctgtttgtggtatagacaaacctatatttaccatgtagattcagaggatttgcatattcataagctt V_(H) Artificial- FIG. 5  59EVKLEESGGGLVQPGGSMKISCVAS Deimmunized deimmunized heavyGFTFSNYWMNWVRQTPEKGLEWV J415-2 chain ALIRSQSNNFATHYAESVKGRVIISR J415-2DDSKSSVYLQMNSLRAEDTAVYYC TRRWNNFWGQGTTVTVSS V_(H) Artificial- FIG. 5  60EVKLEESGGGLVQPGGSMKISCVAS Deimmunized deimmunized heavyGFTFSNYWMNWVRQTPEKGLEWV J415-3 chain AEIRSQSNNFATHYAESVKGRVIISR J415-3DDSKSSVYLQMNSLRAEDTAVYYC TRRWNNFWGQGTTVTVSS J415 V_(H) (DI) Artificial-FIG. 5  61 EVKLEESGGGLVQPGGSMKISCVAS majority  majority sequenceGFTFSNYWMNWVRQTPEKGLEWV sequence AEIRSQSNNFATHYAESVKGRVIISRDDSKSSVYLQMNSLRAEDTAVYYC TRRWNNFWGQGTTVTVSS V_(L) Artificial- FIG. 6  62NIVMTQSPKSMSASAGERMTLTCK Deimmunized deimmunized lightASENVGTYVSWYQQKPTQSPKMLI J415-2 chain YGASNRFTGVPDRFSGSGSGTDFILT J415-2ASSVQAEDPVDYYCGQSYTFPYTFG GGTKLEMK V_(L) Artificial- FIG. 6  63NIVMTQSPKSMSASAGERMTLTCK Deimmunized deimmunized lightASENVGTYVSWYQQKPTQSPKMLI J415-3 chain YGASNRFTGVPDRFSGSGSGTDFILT J415-3ASSVQAEDLVDYYCGQSYTFPYTFG GGTKLEMK V_(L) Artificial- FIG. 6  64NIVMTQSPKSMSASAGERMTLTCK Deimmunized deimmunized lightASENVGTYVSWYQQKPTQSPKMLI J415-4 chain YGASNRFTGVPDRFSGSGSGTDFILT J415-4ISSVQAEDLVDYYCGQSYTFPYTFG GGTKLEMK V_(L) Artificial- FIG. 6  65NIVMTQFPKSMSASAGERMTLTCK Deimmunized deimmunized lightASENVGTYVSWYQQKPEQSPKMLI J415-6 chain YGASNRFTGVPDRFSGSGSGTDFILT J415-6ISSVQAEDLVDYYCGQSYTFPYTFG GGTKLEMK V_(L) Artificial- FIG. 6  66NIVMTQFPKSMSASAGERVTLTCKA Deimmunized deimmunized lightSENVGTYVSWYQQKPTQSPKMLIY J415-7 chain GASNRFTGVPDRFSGSGSGTDFILTI J415-7SSVQAEDLVDYYCGQSYTFPYTFGG GTKLEMK V_(L) Artificial- FIG. 6  67NIVMTQFPKSMSASAGERMTLTCK Deimmunized deimmunized lightASENSGTYVSWYQQKPEQSPKMLI J415-8 chain YGASNRFTGVPDRFSGSGSGTDFILT J415-8ISSVQAEDLVDYYCGQSYTFPYTFG GGTKLEMK J415 V_(L) (DI) Artificial- FIG. 6 68 NIVMTQFPKSMSASAGERMTLTCK majority  majority sequenceASENVGTYVSWYQQKPTQSPKMLI sequence YGASNRFTGVPDRFSGSGSGTDFILTISSVQAEDLVDYYCGQSYTFPYTFG GGTKLEMK MuV_(H) IIIC Mus musculus FIG. 7C  69EVKLEESGGGLVQPGGSMKLSCVA SGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSDNYATHYAESVKGRFTI SRDDSKSSVYLQMNNLRAEDTGIYYCTTGGYGGRRSWFAYWGQGTLV TVSS J415V_(H)/ Artificial- FIG. 7C  70EVKLEESGGGLVQPGGSMKLSCVA MuV_(H) IIIC majority sequenceSGFTFSNYWMNWVRQSPEKGLEW majority  VAEIRLQSDNFATHYAESVKGRVIIS sequenceRDDSKSSVYLQMNNLRAEDTGIYY CTTGGYGGRRSWNAFWGQGTLVT VSS MuV_(L)1Mus musculus FIG. 8C  71 DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQS PKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYS YPLTFGAGTKLELK J415V_(L)/MuV_(L)1 Artificial-FIG. 8C  72 DIVMTQSPSSLAVSAGEKVTLSCKA majority  majority sequenceSESLLNVGNQKTYVAWYQQKPGQS sequence PKLLIYGASTRESGVPDRFTGSGSGTDFILTISSVQAEDLAVYYCGNSYSFP LTFGGGTKLELK J533 V_(H) Mus musculus FIG. 9A 73 gaggtccagctgcagcagtctggacctgagctggttaa CDS (1-354)gcctggggcttcagtgaagatgtcctgcaaggcttctggatacacattcactggctatgttatgcactgggtgaagcagaagcctggacaggtccttgagtggattggatatattaatccttacaatgatgttactaggtataatgggaagttcaaaggcaaggccacactgacctcagacaaatattccagcacagcctacatggagctcagcggcctgacctctgaggactctgcggtctattactgtgcaagaggggagaactggtactactttgactcctggggccgaggcgccactctcacagtctcc tca J533 V_(H) Mus musculusFIG. 9A  74 EVQLQQSGPELVKPGASVKMSCKA (predicted amino SGYTFTGYVMHWVKQKPGQVLEW acid of IGYINPYNDVTRYNGKFKGKATLTS SEQ ID NO: 73)DKYSTAYMELSGLTSEDSAVYYC ARGENWYYFDSWGRGATLTVSS J533 V_(H) Mus musculusFIG. 9A  75 tgaggagactgtgagagtggcgcctcggccccaggag (complementary tcaaagtagtaccagttctcccctcttgcacagtaataga strand of ccgcagagtcctcagaggtcaggccgctgagctccatg SEQ ID NO: 73)taggctgtgctggaatatttgtctgaggtcagtgtggccttgcctttgaacttcccattatacctagtaacatcattgtaaggattaatatatccaatccactcaaggacctgtccaggcttctgcttcacccagtgcataacatagccagtgaatgtgtatccagaagccttgcaggacatcttcactgaagccccaggcttaaccagctcaggtccagactgctgcagctggacctc J533 V_(L) Mus musculus FIG. 10A 76 gacattgtgctgacccaatctccagcttctttggctgtgtc CDS (1-333)tctaggacagagggccaccatatcctgcagagccagtgaaagtattgatagttatgacaatacttttatgcactggtaccagcagaaaccaggacagccacccaacctcctcatctttcgtgcatccatcctagaatctgggatccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaccatttatcctgtggaggctgatgatgttgcaacctattactgtcaccaaagtattgaggatccgtacacgttcggagggggga ccaagctggaaataaaa J533 V_(L)Mus musculus FIG. 10A  77 DIVLTQSPASLAVSLGQRATISCRAS (predicted amino ESIDSYDNTFMHWYQQKPGQPPNL acid of LIFRASILESGIPARFSGSGSGTDFTLSEQ ID NO: 76) TIYPVEADDVATYYCHQSIEDPYTF GGGTKLEIK J533 V_(L)Mus musculus FIG. 10A  78 ttttatttccagcttggtcccccctccgaacgtgtacggat(complementary  cctcaatactttggtgacagtaataggttgcaacatcatc strand of agcctccacaggataaatggtgagggtgaagtctgtcc SEQ ID NO: 76)cagacccactgccactgaacctggcagggatcccagattctaggatggatgcacgaaagatgaggaggttgggtggctgtcctggtttctgctggtaccagtgcataaaagtattgtcataactatcaatactttcactggctctgcaggatatggtggccctctgtcctagagacacagccaaagaagctggag attgggtcagcacaatgtc MuV_(H)IIMus musculus FIG. 9B  79 EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNNWVKQSPGKSLEWI GDINPGNGGTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCA RGYYSSSYMAYYAFDYWGQGTTV TVSSJ533V_(H)/MuV_(H)II Artificial- FIG. 9B  80 EVQLQQSGPELVKPGASVKISCKASmajority  majority sequence GYTFTGYVMNNWVKQSPGQVLEW sequenceIGDINPGNGGTSYNGKFKGKATLTV DKSSSTAYMELSGLTSEDSAVYYCARGENSSSYMAYYAFDSWGQGATVT VSS MuV_(L)-3 Mus musculus FIG. 10B  81DIVLTQSPASLAVSLGQRATISCRAS VESVDSYGNSFMHWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSGTDFT LNIHPVEEDDAATYYCQQSNEDPP WTFGGGTKLEIKJ533V_(L)/MuVL-3 Artificial- FIG. 10B  82 DIVLTQSPASLAVSLGQRATISCRASmajority  majority sequence ESVDSYGNSFMHWYQQKPGQPPNL sequenceLIFAASILESGVPARFSGSGSGTDFTL TIHPVEADDAATYYCQQSIEDPPYTF GGGTKLEfKE99 V_(H) Mus musculus FIG. 11A  83caggtgcagctaaaggagtcaggacctggcctggtgg CDS (1-363)cgtcctcacagagcctgtccatcacatgcaccgtctcaggattctcattaaccgcctatggtattaactgggttcgccagcctccaggaaagggtctggagtggctgggagtgatatggcctgatggaaacacagactataattcaactctcaaatccagactgaacatcttcaaggacaactccaagaaccaagttttcttaaaaatgagcagtttccaaactgatgacacagccagatacttctgtgccagagattegtatggtaacttcaagaggggttggtttgacttctggggccagggcaccactctcac agtctcctca E99 V_(H)Mus musculus FIG. 11A  84 QVQLKESGPGLVASSQSLSITCTVSG (predicted amino FSLTAYGINWVRQPPGKGLEWLGVI acid of WPDGNTDYNSTLKSRLNIFKDNSKNSEQ ID NO: 83) QVFLKMSSFQTDDTARYFCARDSY GNFKRGWFDFWGQGTTLTVSS E99 V_(H)Mus musculus FIG. 11A  85 tgaggagactgtgagagtggtgccctggccccagaagt(complementary  caaaccaacccctcttgaagttaccatacgaatctctggc strand of acagaagtatctggctgtgtcatcagtttggaaactgctc SEQ ID NO: 83)atttttaagaaaacttggttcttggagttgtccttgaagatgttcagtctggatttgagagttgaattatagtctgtgtttccatcaggccatatcactcccagccactccagaccctttcctggaggctggcgaacccagttaataccataggcggttaatgagaatcctgagacggtgcatgtgatggacaggctctgtgaggacgccaccaggccaggtcctgactcctttagct gcacctg E99 V_(L) Mus musculusFIG. 12A  86 aacattgtgatgacccagtctcaaaaattcatgtccacat CDS (1-321)caccaggagacagggtcagggtcacctgcaaggccagtcagaatgtgggttctgatgtagcctggtatcaagcgaaaccaggacaatctcctagaatactgatttactcgacatcctaccgttacagtggggtccctgatcgcttcacagcctatggatctgggacagatttcactctcaccattaccaatgtgcagtctgaagacttgacagagtatttctgtcagcaatataatagctatcctctcacgttcggtgctgggaccaagctggag ctgaaa E99 V_(L) Mus musculusFIG. 12A  87 NIVMTQSQKFMSTSPGDRVRVTCK (predicted amino ASQNVGSDVAWYQAKPGQSPRILIY acid of STSYRYSGVPDRFTAYGSGTDFTLTISEQ ID NO: 86) TNVQSEDLTEYFCQQYNSYPLTFGA GTKLELK E99 V_(L) Mus musculusFIG. 12A  88 tttcagctccagcttggtcccagcaccgaacgtgagag (complementary gatagctattatattgctgacagaaatactctgtcaagtctt strand of cagactgcacattggtaatggtgagagtgaaatctgtcc SEQ ID NO: 86)cagatccataggctgtgaagcgatcagggaccccactgtaacggtaggatgtcgagtaaatcagtattctaggagattgtcctggtttcgcttgataccaggctacatcagaacccacattctgactggccttgcaggtgaccctgaccctgtctcctggtgatgtggacatgaatttttgagactgggtcatcacaa tgtt MuV_(H)IB Mus musculusFIG. 11B  89 QVQLKESGPGLVASSQSLSITCTVSG FSLTAYGINWVRQPPGKGLEWLGVIWPDGNTDYNSTLKSRLN1FKDNSK NQVFLKMSSFQTDDTARYFCARDS YGNFKRGWFDFWGQGTTLTVSSE99VH/MuV_(H)IB Artificial- FIG. 11B  90 QVQLKESGPGLVASSQSLSITCTVSGmajority  majority sequence FSLTAYGINWVRQPPGKGLEWLGVI sequenceWPDGNTDYNSTLKSRLNIFKDNSKN QVFLKMSSFQTDDTARYFCARDSY GNFKRGWFDFWGQGTTLTVSSMuV_(L)-1 Mus musculus FIG. 12B  91 DTVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQ SPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDY SYPLTFGAGTKLELK E99V_(L)/MuV_(L)-1 Artificial-FIG. 12B  92 DIVMTQSQSSLAVSAGDKVTVSCK majority  majority sequenceASQSLLNVGSDKNYVAWYQAKPG sequence QSPKLLIYSASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYFCQNDN SYPLTFGAGTKLELKRA

Humanized or CDR-grafted antibody molecules or immunoglobulins can beproduced by CUR-grafting or CDR substitution, wherein one, two, or allCDRs of an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No.5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; WinterU.S. Pat. No. 5,225,539, the contents of all of which are herebyexpressly incorporated by reference.

Winter describes a CDR-grafting method that may be used to prepare thehumanized antibodies of the present invention (UK Patent Application GB2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), thecontents of which is expressly incorporated by reference. All of theCDRs of a particular human antibody may be replaced with at least aportion of a non-human CDR or only some of the CDRs may be replaced withnon-human CDRs. It is only necessary to replace the number of CDRsrequired for binding of the humanized antibody to a predeterminedantigen.

Humanized antibodies can be generated by replacing sequences of the Fvvariable region that are not directly involved in antigen binding withequivalent sequences from human Fv variable regions. General methods forgenerating humanized antibodies are provided by Morrison, S. L., 1985,Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and byQueen et al. U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761 and U.S.Pat. No. 5,693,762, the contents of all of which are hereby incorporatedby reference. Those methods include isolating, manipulating, andexpressing the nucleic acid sequences that encode all or part ofimmunoglobulin Fv variable regions from at least one of a heavy or lightchain. Sources of such nucleic acid are well known to those skilled inthe art and, for example, may be obtained from a hybridoma producing anantibody against a predetermined target, as described above. Therecombinant DNA encoding the humanized antibody, or fragment thereof,can then be cloned into an appropriate expression vector.

Also within the scope of the invention are humanized antibodies in whichspecific amino acids have been substituted, deleted or added. Inparticular, preferred humanized antibodies have amino acid substitutionsin the framework region, such as to improve binding to the antigen. Forexample, a selected, small number of acceptor framework residues of thehumanized immunoglobulin chain can be replaced by the correspondingdonor amino acids. Preferred locations of the substitutions includeamino acid residues adjacent to the CDR, or which are capable ofinteracting with a CDR (see e.g., U.S. Pat. No. 5,585,089). Criteria forselecting amino acids from the donor are described in U.S. Pat. No.5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contentsof which are hereby incorporated by reference. The acceptor frameworkcan be a mature human antibody framework sequence or a consensussequence.

As used herein, the term “consensus sequence” refers to the sequenceformed from the most frequently occurring amino acids (or nucleotides)in a family of related sequences (See e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987). [In a family ofproteins, each position in the consensus sequence is occupied by theamino acid occurring most frequently at that position in the family. Iftwo amino acids occur equally frequently, either can be included in theconsensus sequence. A “consensus framework” refers to the frameworkregion in the consensus immunoglobulin sequence.

Other techniques for humanizing antibodies are described in Padlan etal. EP 519596 A1, published on Dec. 23, 1992.

The anti-PSMA antibody, or antigen fragment thereof, may also bemodified by specific deletion of human T cell epitopes or“deimmunization” by the methods disclosed in WO 98/52976 and WO00/34317, the contents of which are specifically incorporated byreference herein. Briefly, the murine heavy and light chain variableregions of an anti-PSMA antibody can be analyzed for peptides that bindto MHC Class TT; these peptides represent potential T-cell epitopes (asdefined in WO 98/52976 and WO 00/34317). For detection of potentialT-cell epitopes, a computer modeling approach termed “peptide threading”can be applied, and in addition a database of human MHC class II bindingpeptides can be searched for motifs present in the murine V_(H) andV_(L) sequences, as described in WO 98/52976 and WO 00/34317. Thesemotifs bind to any of the 18 major MHC class II DR allotypes, and thusconstitute potential T cell epitopes. Potential T-cell epitopes detectedcan be eliminated by substituting small numbers of amino acid residuesin the variable regions, or preferably, by single amino acidsubstitutions. As far as possible conservative substitutions are made,often but not exclusively, an amino acid common at this position inhuman germline antibody sequences may be used. Human germline sequencesare disclosed in Tomlinson, I. A. et al. (1992) J. Mol. Biol.227:776-798; Cook, G. P. et al. (1995) Immunol. Today Vol. 16 (5):237-242; Chothia, D. et al. (1992) J. Mol. Bio. 227:799-817. The V BASEdirectory provides a comprehensive directory of human immunoglobulinvariable region sequences (compiled by Tomlinson, I. A. et al. MRCCentre for Protein Engineering, Cambridge, UK). After the deimmunizedV_(H) and V_(L) of an anti-PSMA antibody are constructed by mutagenesisof the murine V_(H) and V_(L) genes. The mutagenized variable sequencecan, optionally, be fused to a human constant region, e.g., human IgG1or κ constant regions.

In some cases a potential T cell epitope will include residues which areknown or predicted to be important for antibody function. For example,potential T cell epitopes are usually biased towards the CDRs. Inaddition, potential T cell epitopes can occur in framework residuesimportant for antibody structure and binding. Changes to eliminate thesepotential epitopes will in some cases require more scrutiny, e.g., bymaking and testing chains with and without the change. Where possible,potential T cell epitopes that overlap the CDRs were eliminated bysubstitutions outside the CDRs. In some cases, an alteration within aCDR is the only option, and thus variants with and without thissubstitution should be tested. In other cases, the substitution requiredto remove a potential T cell epitope is at a residue position within theframework that might be critical for antibody binding. In these cases,variants with and without this substitution should be tested. Thus, insome cases several variant deimmunized heavy and light chain variableregions were designed and various heavy/light chain combinations testedin order to identify the optimal deimmunized antibody. The choice of thefinal deimmunized antibody can then be made by considering the bindingaffinity of the different variants in conjunction with the extent ofdeimmunization, i.e., the number of potential T cell epitopes remainingin the variable region.

The recombinant deimmunized antibody can be transfected into a suitablehost cell for expression, for example, NS0 or CHO cells, to producecomplete recombinant antibodies.

In one embodiment, deimmunized V_(H) and V_(L) of murine J591 regionswere constructed by mutagenesis of the murine V_(H) and V_(L) genes. Themurine J591 variable region sequences are shown in FIGS. 1A-1B.Potential epitopes (identified using a peptide threading program) inmurine J591 heavy chain and light chain variable regions are shown inFIGS. 3A and 3B, respectively. The 13-mer peptides predicted to bind toMHC class II are indicated by the underline, the CDRs are located atresidues 26 to 35, 50 to 66, and 99 to 104 of FIG. 3A and residues 24 to34, 50 to 56, and 89 to 97 of FIG. 3B, and residues altered in thedeimmunized heavy and light chain variable regions are boxed. Wherepossible, amino acid substitutions are those commonly used in humangermline heavy and light chain variable regions. In addition to the insit/co analysis using the peptide threading software, a database ofhuman MHC class II binding peptides was searched for motifs present inthe murine J591 sequence.

The following 13-mers (labeled by first linear residue number of the13-mer) of the murine J591 heavy chain variable region were predicted tobind to MHC Class II were 2, 10, 16, 30, 32, 35, 43, 46, 58, 62, 70, 81,84, 91, and 100 (FIG. 3A). An explanation of the rationale behindchanges made to the residues in the murine J591 heavy chain variableregion is set forth below (note residues altered are identified underthe Kabat numbering system):

-   -   5Q→V removes the potential epitope at residue 2;    -   11,12LV→−VK remove the potential epitope at residue 10;    -   12V→K is also changed to remove a motif from the database of        human MHC class II binding peptides;    -   16,17TS→AT, and 19R→K remove the potential epitope at residue        16;    -   the epitope at residue 30 spans CDR1 and is therefore unaltered;    -   40,41SH→AP removes potential epitopes at residues 32 and 35;    -   44S→G reduces binding score for epitope at 43, this 13 mer spans        CDR2;    -   the epitopes at residues 46, 58 and 62 span CDR2, and are thus        unaltered;    -   75,76SS→TD remove the potential epitope at residue 70;    -   82aR→S, 83T→R remove potential epitopes at residues 81 and 84;    -   87S→T this change made to remove a motif from the database of        human MHC class II binding peptides;    -   the epitope at residue 91 spans CDR3 and is therefore unaltered;        and    -   108T→L removes the potential epitope at residue 100.

The following 13-mers (labeled by first linear residue number of the13-mer) of the murine J591 light chain variable region that werepredicted to bind to MHC Class II molecules were 1, 8, 17, 27, 30, 31,35, 45, 47, 56, 60, 71, 73, 81, 94 (FIG. 3B). An explanation of therationale behind changes made to the residues in the murine J591 lightchain variable region is set forth below (note residues altered areidentified under the Kabat numbering system):

-   -   3V→Q removes potential epitope at residue 1;    -   8-11HKFM→PSSL removes potential epitope at residue 8(13);    -   20-22SII→TLT removes potential epitopes at residues 17 and 20;    -   21I→L is also changed to remove a motif from the database of        human MHC class II binding peptides;    -   the epitope at residue 27 spans CDR1 and is therefore unaltered;    -   42Q→P reduces the binding score for the epitope at residue 31;    -   the epitopes at residues 44 and 47 span CDR2 and are thus        unaltered;    -   58V→I is changed to remove a motif from the database of human        MHC class II binding peptides;    -   60D→S, 62T→S removes the epitopes at residues 56 and 60;    -   76-78TNV→SSL, 80S→P, 83L→F removes the epitopes at residues 71,        73, 76, and 81;    -   87F→YI is changed to remove a motif from the database of human        MHC class II binding peptides;    -   100 A→P and 103 M→K remove the epitope at residue 94; and    -   104 L→V and 106 L→I are changed to remove a motif from the        database of human MHC class II binding peptides.

The amino acid and nucleotide sequences for the deimmunized J591 heavyand light chain variable regions are shown in FIGS. 2A-2B and 4A-4B,respectively (see also Table 8).

Human IgG1 or κ constant regions were added and the composite genestransfected into NS0 cells to produce complete recombinant anti-PSMAantibodies. These antibodies bound to PSMA (on LNCap cells) asefficiently as the original murine antibody, and have reduced or noimmunogenicity in man.

The design of deimmunized J415 was similar to the making of thedeimmunized J591 antibody. The heavy and light chain sequences werecloned from the hybridoma designated HB-12109. These sequences werecloned, sequenced and expressed as a chimeric antibody for use as acontrol antibody. The murine V region sequences were subjected topeptide threading to identify potential T cell epitopes, throughanalysis of binding to 18 different human MHC class II allotypes. Theresults of the peptide threading analysis for the murine sequences areshown in Table 9.

TABLE 9 Potential T cell epitopes in murine J415 sequences Number ofpotential Location of potential epitopes+ (no. of Sequence T cellpotential MHC binders from 18 groups tested) Murine 12 10(17), 16(13),21(9), 30(6), 35(16), 43(8), 46(6), J415 V_(H) 49(8), 64(6), 80(15),86(15), 104(6) Murine 13 5(5), 11(18), 13(11), 17(5), 27(8), 31(7),56(15), J415 V_(K) 60(12), 70(5), 71(11), 73(17), 76(7), 81(17) +firstamino acid of potential epitope, numbering E or N amino acid number 1 toS or K amino acid number 107 and 116 for V_(H) and V_(K) respectively.

Primary deimmunized V_(H) and V_(L) sequences were defined (J415D1VH1,J415D1VK1). As generation of the primary deimmunized sequences requiresa small number of amino acid substitutions that might affect the bindingof the final deimmunized molecule, three other variant V_(H)S and sevenother V_(L)S were designed (see FIGS. 5 and 6). The nucleotide sequencesfor the primary deimmunized V_(H) and V_(L), regions are shown in FIGS.7A and 8A, respectively. Comparisons of the amino acid sequences of themurine and deimmunized V regions of J415 are shown in FIG. 5 for V_(H)and FIG. 6 for V_(L).

An explanation of the rational behind some of the changes made to theresidues in the murine J415 heavy chain variable region is set forthbelow (note residues altered are identified according to the linearnumbering shown in FIG. 5):

-   -   20L→I removes the potential epitope at residues 10 and 16;    -   87N→S removes the potential epitopes at residues 80 and 86;    -   94,95GI→AV remove the potential epitope at residue 86; and    -   112L→V removes the potential epitope at residue 104.

An explanation of the rational behind some of the changes made to theresidues in the murine J415 light chain variable region is set forthbelow (note residues altered are identified according to the linearnumbering shown in FIG. 6):

-   -   131A removes the potential epitopes at residues 5, 11 and 13;    -   15V A removes the potential epitopes at residues 5, 11, and 13;    -   19V-M removes the potential epitopes at residues 11, 13, and 17;    -   41E-T removes the potential epitope at residue 31;    -   63T-S removes the potential epitopes at residues 56 and 60;    -   68A-G removes the potential epitopes at residues 56 and 60 and    -   80T-A removes the potential epitopes at residues 70, 71, 73, and        76;

The deimmunized variable regions for J415 were constructed by the methodof overlapping PCR recombination. The cloned murine V_(H) and V_(K)genes were used as templates for mutagenesis of the framework regions tothe required deimmunized sequences. Sets of mutagenic primer pairs weresynthesized encompassing the regions to be altered. The vectors VH-PCR1and VK-PCR1 (Riechmann et al. (1988) Nature 332:323-7) were used astemplates to introduce 5′ flanking sequence including the leader signalpeptide, leader intron and the murine immunoglobulin promoter, and 3′flanking sequence including the splice site, and intron sequences. Thedeimmunized V regions produced were cloned into pUC19 and the entire DNAsequence was confirmed to be correct for each deimmunized V_(H) andV_(L).

The deimmunized heavy and light chain V-region genes were excised frompUC19 as HindIII to BamHI fragments, which include the murine heavychain immunoglobulin promoter, the leader signal peptide, leader intron,the V_(H) or V_(L) sequence and the splice site. These were transferredto the expression vectors pSVgpt and pSVhyg, which include human IgG1 orκ constant regions respectively and markers for selection in mammaliancells. The DNA sequence was confirmed to be correct for the deimmunizedV_(H) and V_(L) in the expression vectors.

For the transfection of expression vectors pSVgpt1415VHHuIgG1 andpSVhygJ415VKHuCK into NS0 (a non-immunoglobulin producing mouse myeloma,obtained from the European Collection of Animal Cell Cultures, Porton UK(ECACC No. 85110503)) cells, 3 and 6 ug of plasmid DNA respectively wasprepared and then linearized with Pvul to improve transfectionefficiency. The ethanol precipitated DNA was then mixed with asemi-confluent flask of NS0 cells that had been harvested bycentrifugation and resuspended in 0.5 ml of non-selective Dulbecco'sModified Eagle's Medium (DMEM) (Life Technologies Inc.) in a 0.4 cm genepulser cuvette. The cells and DNA were chilled on ice for 5 minutesbefore a pulse of 170V, 960 μF was applied. The cuvette was returned toice for a further 20 minutes before being transferred to a 75 cm² flaskcontaining 20 mls non-selective DMEM to recover for 24 hours. The cellswere then harvested and resuspended in selective DMEM and plated over4×96 well plates, 200 μl/well. A similar protocol was followed for thetransfection of expression vectors encoding the deJ591 antibody heavychain and light chain subunits into NS0 cells.

To culture, select, and expand NS0 cell lines, the cells are grown at37° C., 5% C0₂ and 10% FBS. To prepare non-selective medium for routineculture of NS0 cells, the culture medium is Dulbecco's Modification ofEagle's Medium (DMEM)(Life Technologies, Catalogue No: 31965-023)supplemented with 10% fetal bovine serum of USA origin (LifeTechnologies, Fetal Bovine Serum Cat No: 16000), Antibiotic/Antimycoticsolution (Life Technologies, Cat No: 15240), Gentamycin (LifeTechnologies, catalogue No: 15710), Sodium pyruvate (Life Technologies,Catalogue No: 11360-039). When growing NS0 cells up to saturation forantibody production do not add the xanthine and mycophenolic acid andthe FBS is reduced to 5%.

To prepare selective medium for culture of NS0 transfectomas, theculture medium is Dulbecco's Modification of Eagle's Medium (DMEM)(LifeTechnologies, Catalogue No: 31965-023) supplemented with 10% fetalbovine serum of USA origin (Life Technologies, Fetal Bovine Serum CatNo: 16000), Antibiotic/Antimycotic solution (Life Technologies, Cat No:15240), Gentamycin (Life Technologies, catalogue No: 15710), Sodiumpyruvate (Life Technologies, Catalogue No: 11360-039), 250 μg/mlxanthine (Sigma Catalogue No: X-3627, stock made up at 25 mg/ml in 0.5MNaOH), and 0.8 μg/ml mycophenolic acid (Sigma Catalogue No: M-3536,stock made up at 2.5 mg/ml in 100% ethanol).

After approximately 10 days the cell colonies expressing the gpt genewere visible to the naked eye. The plates were then screened forantibody production using the following protocol for human IgG1/κScreening ELISA. 6 single colonies were picked from wells with high ODsgreater than 0.7 into a 24 well cell culture plate. Within 5-6 days thecells were expanded into a 25 cm² flask. The antibody productivity ofthe selected clones was assayed using the following protocol for humanIgG1/κ ELISA from saturated cultures in the 24 well and 25 cm² flasks.

The details of the protocol are as follows. ELISA plates (DynatechImmulon 2) are coated with 100 μL per well with sheep α human κ antibody(The Binding Site Cat No: AU015) diluted 1:1000 in carbonate/bicarbonatecoating buffer pH9.6 (Sigma Cat: C-3041). The coated plate is incubatedat 4° C. overnight or 1 hr at 37° C. The plate is then washed 3 timeswith PBST (PBS with 0.05% Tween 20). The samples are added, 100 μL perwell from 24 well plates, 25 μL+75 μL PBST for 96 well plates. Blankwells are treated with PBST only. The reaction mixture is incubated atroom temperature for 1 hr. Then, the plate is wash 3 times with PBST(PBS with 0.05% Tween 20). The secondary antibody, peroxidase conjugatedsheep α human IgG γ chain specific is added (The Binding Site Cat No:APO04) at a ratio of 1:1000 in PBST, 100 μL per well. The mixture isincubated at room temperature for 1 hour. The mixture is then washed 3times with PBST (PBS with 0.05% Tween 20).

To make up the substrate, one tablet (20 mg) of OPD (o-PHENYLENEDIAMINE) (Sigma Cat No: P-7288) is dissolved in 45 ml of H₂O plus 5 ml10× peroxidase buffer (make 10× peroxidase buffer with Sigma phosphatecitrate buffer tablets pH 5.0, P-4809), add 10 μL 30% (w/w) hydrogenperoxide (Sigma Cat No: H1109) just before use. The substrate is thenadded at 100 μL per well and incubate RT for 5 min or as required. Whenthe color develops, the reaction can be stopped by adding 25 μL 12.5%H2SO4. The results are read at 492 nm.

Expression and Expansion of J591 and J415 Deimmunized Antibodies

The clones with the highest productivity were expanded into a 75 cm²flask and then into 2×175 cm² flasks. The cells from one of the 175 cm²flask was used to inoculate 4× triple layer flasks (500 cm², Nalge NuncInternational) containing non selective DMEM containing 5% FBS, cellsfrom the other were frozen as detailed in the protocol for freezing NS0cells detailed below.

To cryoprotect mammalian cells and resurrect cells from liquid nitrogen,the following materials are needed: Fetal Bovine serum (LifeTechnologies Cat No: 16000), DMSO (Sigma, Cat No: D4540), 2 ml cryotubes(Nunc or Greiner), and polystyrene box with walls 1-2 cm thick. Briefly,actively growing cells are harvested by centrifugation (1000 rpm, 5 min)and resuspended at about 10⁷ cells/ml in 10% DMSO/90% FBS. As a roughguide, cells grown to a semi-confluency should be resuspended in 1 mlfor a 75 cm² flask or 2.5 ml for a 175 cm² flask. A required number oftubes are cooled and labeled in ice. 1 ml portions are aliquoted tolabeled cryotubes. The cryotubes are placed in polystyrene box at −70°C. for at least 4 h, or overnight. The vials are transferred to canesand place in liquid nitrogen. A record of the storage should be madeboth in the canister index and the central cell line indexing system.

To thaw the cells from liquid nitrogen, the vial is removed from liquidnitrogen and contents are thawed quickly by incubation at 37° C., whileswirling in a waterbath. The outside of the vial is cleaned with 70%methylated spirits. The suspension is transferred to a universalcontainer. 10 ml of the medium to be used to propagate the cell line isadded dropwise, swirling to mix. The cells are harvested bycentrifugation (1000 rpm, 5 min). The supernatant is discarded. Thecells are resuspended in 20 ml growth medium and transfer to a 75 cm²flask. If low viability is suspected, extra serum can be added to thegrowth medium to 20%, use only 5 ml, and transfer to a 25 cm² flask.

After 10-14 days the 500 ml to 1 liter static saturated cultures wereharvested. Antibody was purified, by ProSepA (Millipore Ltd.) affinitychromatography using the following protocol for antibody purification.The purified antibody preparation was sterilized by filtration andstored at 4° C.

The antibody purification protocol is as follows: NS0 transfectoma cellline producing antibody is grown in DMEM 5% FCS in Nunc Triple layerflasks, 250 ml per flask (total volume 1 L) for 10-14 days until nearingsaturation. Conditioned medium collected and spun at 3000 rpm for 5 minin bench centrifuge 5 mins to remove cells. 1/10^(th) volume 1M Tris-HClpH8 (Sigma Cat: T3038) is then added to cell supernatant to make this0.1 M Tris-HCl pH8. 0.5 to 1 ml Prosep A (Millipore Cat: 113 111824) isadded and stirred overnight at room temperature. Prosep A collected byspinning at 3000 rpm for 5 mins then packed into a Biorad Poly-Prepcolumn (Cat: 73 1-1550). The column is washed with 10 ml PBS, theneluted in 1 ml fractions with 0.1M Glycine pH 3.0. Each fraction iscollected into a tube containing 100 microL 1M Tris-HCl pH 8 (Sigma, asabove). Absorbance of each fraction is measured at 280 nm. Fractionscontaining the antibody are pooled and dialyzed against PBS overnight atroom temperature. The preparation is sterilized by filtration through a0.2 micron syringe filter and the absorbance of each fraction ismeasured at 280 nm. The antibody concentration is determined by ELISAfor human IgG.

The purified antibody can be quantified using the protocol for HumanIgG1/κ ELISA described above.

Testing of J415 Deimmunized Antibodies

The J415 deimmunized antibodies (including various combinations of thedeimmunized light chain and heavy chain subunits) were tested in anELISA for binding to LNCap membrane preparation following the protocolas detailed above. ELISA plates were coated with LNCap membranepreparation and blocked with 5% BSA in phosphate buffered saline.Doubling dilutions of the J415 chimeric antibody (murine variable heavyand light chain regions fused to human constant heavy and light chainregions, respectively) and the deimmunized antibodies were applied.Detection was with horseradish peroxidase conjugated goat anti-human IgGand donkey anti-mouse for chimeric and mouse antibodies respectively.Color was developed with o-phenylene diamine substrate.

The antibody composed of deimmunized J415 heavy chain version 4 combinedwith deimmunized J415 light chain version 5 shows equivalent binding toLNCap cells as compared to the chimeric antibody. Also, when DIVK5 iscombined with heavy chain versions 1 and 2, binding to LNCap cells isequivalent to that of the chimeric antibody when tissue culturesupernatant is analyzed. These data can be confirmed with purifiedantibody. When light chains 1, 2, 3 were combined with any of the J415heavy chain versions 1, 2, 3, and 4 no antibody was produced.Deimmunized J415 light chain versions 1, 2, and 3 may be defective onstructural grounds. The best chain combination for higher affinity anddecreased immunogenicity is D1VH4/DIVK5.

The antibody composed of deimmunized heavy chain version 4 combined withdeimmunized light chain version 5 showed equivalent binding to LNCapcompared to the chimeric antibody. Also, when DIVK5 is combined withheavy chain versions 1 and 2, binding to LNCap cells is two-fold lessthan that of the chimeric when purified antibody is analyzed.

Monoclonal anti-PSMA antibodies can also be generated by other methodsknown to those skilled in the art of recombinant DNA technology.

Anti-PSMA antibodies that are not intact antibodies are also useful inthis invention. Such antibodies may be derived from any of theantibodies described above. For example, antigen-binding fragments, aswell as full-length monomeric, dimeric or trimeric polypeptides derivedfrom the above-described antibodies are themselves useful. Usefulantibody homologs of this type include (i) a Fab fragment, a monovalentfragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment consisting ofthe VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VHdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,(1989) Nature 341:544-546), which consists of a VH domain; and (vi) anisolated complementarity determining region (CDR), e.g., one or moreisolated CDRs together with sufficient framework to provide an antigenbinding fragment. Furthermore, although the two domains of the Fvfragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare also intended to be encompassed within the term “antigen-bindingfragment” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

Antibody fragments may also be produced by chemical methods, e.g., bycleaving an intact antibody with a protease, such as pepsin or papain,and optionally treating the cleaved product with a reducing agent.Alternatively, useful fragments may be produced by using host cellstransformed with truncated heavy and/or light chain genes.

Monoclonal, chimeric, humanized, deimmunized antibodies, which have beenmodified by, e.g., deleting, adding, or substituting other portions ofthe antibody, e.g., the constant region, are also within the scope ofthe invention. For example, an antibody can be modified as follows: (i)by deleting the constant region; (ii) by replacing the constant regionwith another constant region, e.g., a constant region meant to increasehalf-life, stability or affinity of the antibody, or a constant regionfrom another species or antibody class; or (iii) by modifying one ormore amino acids in the constant region to alter, for example, thenumber of glycosylation sites, effector cell function, Fc receptor (FcR)binding, complement fixation, among others.

In one embodiment, the constant region of the antibody can be replacedby another constant region from, e.g., a different species. Thisreplacement can be carried out using molecular biology techniques. Forexample, the nucleic acid encoding the VL or VH region of a antibody canbe converted to a full-length light or heavy chain gene, respectively,by operatively linking the VH or VL-encoding nucleic acid to anothernucleic acid encoding the light or heavy chain constant regions. Thesequences of human light and heavy chain constant region genes are knownin the art. Preferably, the constant region is human, but constantspecies from other species, e.g., rodent (e.g., mouse or rat), primate,camel, rabbit can also be used. Constant regions from these species areknown in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242).

Methods for altering an antibody constant region are known in the art.Antibodies with altered function, e.g. altered affinity for an effectorligand, such as FcR on a cell, or the C1 component of complement can beproduced by replacing at least one amino acid residue in the constantportion of the antibody with a different residue (see e.g., EP 388,151A1, U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, the contents ofall of which are hereby incorporated by reference). Similar type ofalterations could be described which if applied to the murine, or otherspecies immunoglobulin would reduce or eliminate these functions.

An anti-PSMA antibody, or antigen-binding fragment thereof, can bederivatized or linked to another functional molecule (e.g., anotherpeptide or protein). Accordingly, the antibodies and antibody portionsof the invention are intended to include derivatized and otherwisemodified forms of the antibodies described herein, includingimmunoadhesion molecules. For example, an antibody or antibody portionof the invention can be functionally linked (by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), a detectable agent, a cytotoxic agent, apharmaceutical agent, and/or a protein or peptide that can mediateassociation of the antibody or antibody portion with another molecule(such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Useful detectable agents with which an antibody or antibody portion ofthe invention may be derivatized (or labeled) to include fluorescentcompounds, various enzymes, prosthetic groups, luminescent materials,bioluminescent materials, fluorescent emitting metal atoms, e.g.,europium (Eu), and other lanthanides, and radioactive materials(described below). Exemplary fluorescent detectable agents includefluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and thelike. An antibody may also be derivatized with detectable enzymes, suchas alkaline phosphatase, horseradish peroxidase, β-galactosidase,acetylcholinesterase, glucose oxidase and the like. When an antibody isderivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with a prosthetic group(e.g., streptavidin/biotin and avidin/biotin). For example, an antibodymay be derivatized with biotin, and detected through indirectmeasurement of avidin or streptavidin binding. Examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of bioluminescent materials include luciferase,luciferin, and aequorin.

Labeled antibodies can be used, for example, diagnostically and/orexperimentally in a number of contexts, including (i) to isolate apredetermined antigen by standard techniques, such as affinitychromatography or immunoprecipitation; (ii) to detect a predeterminedantigen (e.g., in a cellular lysate or cell supernatant) in order toevaluate the abundance and pattern of expression of the protein; (iii)to monitor protein levels in tissue as part of a clinical testingprocedure, e.g., to determine the efficacy of a given treatment regimen.

An anti-PSMA antibody or antigen-binding fragment thereof may beconjugated to a another molecular entity, typically a label or atherapeutic (e.g., a cytotoxic or cytostatic) agent or moiety.

Radioactive isotopes can be used in diagnostic or therapeuticapplications. Radioactive isotopes that can be coupled to the anti-PSMAantibodies include, but are not limited to α-, β-, or γ-emitters, or β-and γ-emitters. Such radioactive isotopes include, but are not limitedto iodine (¹³¹I or ¹²⁵I), yttrium (⁹⁰Y), lutetium (¹⁷⁷Lu), actinium(²²⁵Ac), praseodymium, astatine (²¹¹At) rhenium (¹⁸⁶Re), bismuth (²¹²Bior ²¹³Bi), indium (¹¹¹In), technetium (⁹⁹mTc), phosphorus (³²P), rhodium(¹⁸⁸Rh) sulfur (³⁵S), carbon (¹⁴C), tritium (³H), chromium (⁵¹Cr),chlorine (³⁶Cl), cobalt (⁵⁷Co or ⁵⁸Co), iron (⁵⁹Fe), selenium (⁷⁵Se), orgallium (⁶⁷Ga). Radioisotopes useful as therapeutic agents includeyttrium (⁹⁰Y), lutetium (¹⁷⁷Lu), actinium (²²⁵Ac), praseodymium,astatine (²¹¹At) rhenium (¹⁸⁶Re), bismuth (²¹²Bi and ²¹³Bi), and rhodium(¹⁸⁸Rh). Radioisotopes useful as labels, e.g., for use in diagnostics,include iodine (¹³¹I or ¹²⁵I), indium (¹¹¹In), technetium (⁹⁹mTc),phosphorus (³²P), carbon (¹⁴C), and tritium (³H), or one or more of thetherapeutic isotopes listed above.

The invention provides radiolabeled anti-PSMA antibodies and methods oflabeling the same. In one embodiment, a method of labeling an anti-PSMAantibody is disclosed. The method includes contacting an anti-PSMAantibody, e.g. an anti-PSMA antibody described herein, with a chelatingagent, e.g., 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid(DOTA), to thereby produced a conjugated antibody. The conjugatedantibody is radiolabeled with a radioisotope, e.g., ¹¹¹Indium, ⁹⁰Yttriumand ¹⁷⁷Lutetium, to thereby produce a labeled anti-PSMA antibody.Detailed procedures for radiolabeling an anti-PSMA antibody aredescribed in more detail in the sections below and the appendedexamples. For example, the anti-PSMA antibodies can be radiolabeled with¹¹¹Indium, ⁹⁰Yttrium, or ¹⁷⁷Lutetium by coupling with1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) asdescribed in U.S. Ser. No. 60/295,214, filed on Jun. 1, 2001, thecontents of which are incorporated by reference in its entirety.Detailed experimental protocols for chelating anti-PSMA antibodies aredescribed in Example 16 of U.S. Ser. No. 60/295,214, which isspecifically incorporated by reference in the present application and isreproduced below as Example 1. Where DOTA is used as a chelating agent,to obtain a consistent conjugation ratio between DOTA and anti-PSMAantibody and thus control the quality of the final product, theconcentration/amount of reactive DOTA-NHS ester present in the reactionmixture can be determined using methods known in the art, including themethods described herein in Example, e.g., using LC and MS.

As is discussed above the antibody can be conjugated to a therapeuticagent. Therapeutically active radioisotopes have already been mentioned.Examples of other therapeutic agents include TAXOL® (paclitaxel),cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D,1-dihydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids,e.g., maytansinol (sec U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat.Nos. 5,475,092, 5,585,499, 5,846,545) and analogs or homologs thereof.Therapeutic agents include, but are not limited to, antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (11) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, TAXOL® (paclitaxel) and maytansinoids).

The conjugates of the invention can be used for modifying a givenbiological response. The therapeutic agent is not to be construed aslimited to classical chemical therapeutic agents. For example, thetherapeutic agent may be a protein or polypeptide possessing a desiredbiological activity. Such proteins may include, for example, a toxinsuch as abrin, ricin A, pseudomonas exotoxin, diphtheria toxin, or acomponent thereof (e.g., a component of pseudomonas exotoxin is PE38); aprotein such as tumor necrosis factor, interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator; or,biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors. Similarly,the therapeutic agent can be a viral particle, e.g., a recombinant viralparticle, that is conjugated (e.g., via a chemical linker) or fused(e.g., via a viral coat protein) to an anti-PSMA antibody of theinvention. Introduction of the viral nucleic acid molecules, e.g.,recombinant viral nucleic acid molecules, into cells, e.g., prostatecancer cells or vascular endothelial cells associated with tumors, thatexpress PSMA can occur following binding and endocytosis of theanti-PSMA antibody/viral particle conjugate or fusion.

Nucleic Acids, Vectors and Host Cells

Another aspect of the invention pertains to isolated nucleic acid,vector and host cell compositions that can be used for recombinantexpression of the modified antibodies and antigen-binding fragment ofthe invention. In one embodiment, a first and second isolated nucleicacid comprising a nucleotide sequence encoding heavy and light chainvariable regions, respectively, of an anti-PSMA antibody, e.g., amodified anti-PSMA antibody (e.g., a deimmunized J591 or J415 anti-PSMAantibody), or an antigen fragment thereof, are provided.

The nucleotide and amino acid sequence of the modified (deimmunized)anti-PSMA J591 immunoglobulin light chain variable region is shown inFIG. 4B (SEQ ID NO:25 and 22, respectively). The non-codingcomplementary nucleotide sequence is also shown in FIG. 4B (SEQ IDNO:26). The J591 deimmunized anti-PSMA antibody light chain variableregion contains the following regions: an FR1 domain corresponding toabout amino acid residues 1-23 of SEQ ID NO:22 (linear numbering; seealso SEQ ID NO:13), which is encoded by about nucleotides 261-329 of SEQID NO:25; a CDR1 domain corresponding to about amino acid residues 24-34of SEQ ID NO:22 (linear numbering; see also SEQ ID NO:4), which isencoded by about nucleotides 330-362 of SEQ ID NO:25; an FR2 domaincorresponding to about amino acid residues 35-49 of SEQ ID NO:22 (linearnumbering; see also SEQ ID NO:14), which is encoded by about nucleotides363-407 of SEQ ID NO:25; a CDR2 domain corresponding to about amino acidresidues 50-56 of SEQ ID NO:22 (linear numbering; see SEQ ID NO:5),which is encoded by about nucleotides 408-428 of SEQ ID NO:25; an FR3domain corresponding to about amino acid residues 57-88 of SEQ ID NO:22(linear numbering; see also SEQ ID NO:15), which is encoded by aboutnucleotides 429-524 of SEQ ID NO:25; a CDR3 domain corresponding toabout amino acid residues 89-97 of SEQ ID NO:22 (linear numbering; seealso SEQ ID NO:6), which is encoded by about nucleotides 525-551 of SEQID NO:25; and an FR4 domain corresponding to about amino acid residues98-107 of SEQ ID NO:22 (linear numbering; see also SEQ ID NO:16), whichis encoded by about nucleotides 552-581 of SEQ ID NO:25.

The nucleotide and amino acid sequence of the modified (deimmunized)anti-PSMA J591 immunoglobulin heavy chain variable region is shown inFIG. 4A (SEQ ID NO:23 and 21, respectively). The non-codingcomplementary sequence is also shown in FIG. 4A (SEQ ID NO:24). The J591deimmunized anti-PSMA antibody heavy chain variable region contains thefollowing regions: an FR1 domain corresponding to about amino acidresidues 1-25 of SEQ ID NO:21 (linear numbering; see also SEQ ID NO:9),which is encoded by about nucleotides 261-335 of SEQ ID NO:23; a CDR1domain corresponding to about amino acid residues 26-35 of SEQ ID NO:21(linear numbering; see also SEQ ID NO:1), which is encoded by aboutnucleotides 336-365 of SEQ ID NO:23; an FR2 domain corresponding toabout amino acid residues 36-49 of SEQ ID NO:21 (linear numbering; seealso SEQ ID NO:10), which is encoded by about nucleotides 366-407 of SEQID NO:23; a CDR2 domain of corresponding to about amino acid residues50-66 of SEQ ID NO:21 (linear numbering; see also SEQ ID NO:2), which isencoded by about nucleotides 408-458 of SEQ ID NO:23; an FR3 domaincorresponding to about amino acid residues 67-98 of SEQ ID NO:21 (linearnumbering; see also SEQ ID NO:11), which is encoded by about nucleotides459-554 of SEQ ID NO:23; a CDR3 domain corresponding to about amino acidresidues 99-104 of SEQ ID NO:21 (linear numbering; see also SEQ IDNO:3), which is encoded by about nucleotides 555-572 of SEQ ID NO:23;and an FR4 domain corresponding to about amino acid residues 105-115 ofSEQ ID NO:21 (linear numbering; see also SEQ ID NO:9), which is encodedby about nucleotides 573-605 of SEQ ID NO:23.

The nucleotide and amino acid sequence of the modified (deimmunized)anti-PSMA J415 immunoglobulin light chain variable region (J415DIVK1) isshown in FIG. 8A (SEQ ID NO:56 and 57, respectively). The non-codingcomplementary nucleotide sequence of J415DIVK1 is also shown in FIG. 8A(SEQ ID NO:58). The J415 deimmunized anti-PSMA antibody light chainvariable region contains the following regions: an FR1 domaincorresponding to about amino acid residues 1-23 of SEQ ID NO:57 (linearnumbering; see also SEQ ID NO:41), which is encoded by about nucleotides261-329 of SEQ ID NO:56; a CDR1 domain corresponding to about amino acidresidues 24-34 of SEQ ID NO:57 (linear numbering; see also SEQ IDNO:32), which is encoded by about nucleotides 330-362 of SEQ ID NO:56;an FR2 domain corresponding to about amino acid residues 35-49 of SEQ IDNO:57 (linear numbering; see also SEQ ID NO:42), which is encoded byabout nucleotides 363-407 of SEQ ID NO:56; a CDR2 domain correspondingto about amino acid residues 50-56 of SEQ ID NO:57 (linear numbering;see also SEQ ID NO:33), which is encoded by about nucleotides 408-428 ofSEQ ID NO:56; an FR3 domain corresponding to about amino acid residues57-88 of SEQ ID NO:57 (linear numbering; see also SEQ ID NO:43), whichis encoded by about nucleotides 429-524 of SEQ ID NO:56; a CDR3 domaincorresponding to about amino acid residues 89-97 of SEQ TD NO:57 (linearnumbering; see also SEQ ID NO:34), which is encoded by about nucleotides525-551 of SEQ ID NO:56; and an FR4 domain corresponding to about aminoacid residues 98-107 of SEQ ID NO:57 (linear numbering; see also SEQ IDNO:44), which is encoded by about nucleotides 552-581 of SEQ ID NO:56.The nucleotide and amino acid sequences of the preferred modified(deimmunized) anti-PSMA J415 immunoglobulin light chain variable region(J415DIVK5) are shown in SEQ ID NO:50 and 52, respectively; J415DIVK5can be broken down into its component sequences in a manner identical tothat shown above for J415DIVK1.

The nucleotide and amino acid sequence of the modified (deimmunized)anti-PSMA J415 immunoglobulin heavy chain variable region is shown inFIG. 7A (SEQ ID NO:53 and 54, respectively). The non-codingcomplementary sequence is also shown in FIG. 7A (SEQ ID NO:55). The J415deimmunized anti-PSMA antibody heavy chain variable region contains thefollowing regions: an FR1 domain corresponding to about amino acidresidues 1-25 of SEQ ID NO:54 (linear numbering; see also SEQ ID NO:37),which is encoded by about nucleotides 261-335 of SEQ ID NO:53; a CDR1domain corresponding to about amino acid residues 26-35 of SEQ ID NO:54(linear numbering; see also SEQ ID NO:29), which is encoded by aboutnucleotides 336-365 of SEQ ID NO:53; an FR2 domain corresponding toabout amino acid residues 36-49 of SEQ ID NO:54 (linear numbering; seealso SEQ ID NO:38), which is encoded by about nucleotides 366-407 of SEQID NO:53; a CDR2 domain corresponding to about amino acid residues 50-68of SEQ ID NO:54 (linear numbering; see also SEQ ID NO:30), which isencoded by about nucleotides 408-464 of SEQ ID NO:53; an FR3 domaincorresponding to about amino acid residues 69-100 of SEQ ID NO:54(linear numbering; see also SEQ ID NO:39), which is encoded by aboutnucleotides 465-560 of SEQ ID NO:53; a CDR3 domain corresponding toabout amino acid residues 101-105 of SEQ ID NO:54 (linear numbering; seealso SEQ ID NO:31), which is encoded by about nucleotides 561-575 of SEQID NO:53; and an FR4 domain corresponding to about amino acid residues106-116 of SEQ ID NO:54 (linear numbering; see also SEQ ID NO:40), whichis encoded by about nucleotides 576-608 of SEQ ID NO:53. The nucleotideand amino acid sequences of the preferred modified (deimmunized)anti-PSMA J415 immunoglobulin heavy chain variable region (J415DIVH4)are shown in SEQ ID NO:51 and 49, respectively; J415DIVH4 can be brokendown into its component sequences in a manner identical to that shownabove for J415DIVH1.

It will be appreciated by the skilled artisan that nucleotide sequencesencoding anti-PSMA modified antibodies (e.g., FR domains, e.g., FR1-4),can be derived from the nucleotide and amino acid sequences described inthe present application using the genetic code and standard molecularbiology techniques.

In one embodiment, the isolated nucleic acid comprises an anti-PSMAmodified antibody heavy chain variable region nucleotide sequence havinga nucleotide sequence as shown in FIG. 4A (SEQ ID NO:23), FIG. 7A (SEQID NO:53) or SEQ ID NO:51 (for J415DIVH4) or a complement thereof (e.g.,SEQ ID NO:24 or SEQ ID NO:55), the nucleotide sequence of the heavychain variable region of the antibody produced by the NS0 cell linehaving ATCC Accession Number PTA-3709 or PTA-4174 or a complementthereof; a sequence at least 85%, 90%, 95%, 99% or more identitythereto; or a sequence capable of hybridizing under stringent conditionsdescribed herein (e.g., highly stringent conditions) to a nucleotidesequence shown in FIG. 4A (SEQ ID NO:23), FIG. 7A (SEQ ID NO:53), SEQ IDNO:51, or a complement thereof (e.g., SEQ ID NO:24 or SEQ ID NO:55), orthe nucleotide sequence of the heavy chain variable region of theantibody produced by the NS0 cell line having ATCC Accession NumberPTA-3709 or PTA-4174, or a complement thereof.

In another embodiment, the isolated nucleic acid encodes an anti-PSMAmodified antibody heavy chain variable region amino acid sequence havingan amino acid sequence as shown in FIG. 2A (SEQ ID NO:21) or FIG. 5(e.g., SEQ ID NO:49), or the amino acid sequence of the heavy chainvariable region of the antibody produced by the NS0 cell line havingATCC Accession Number PTA-3709 or PTA-4174; a sequence at least 85%,90%, 95%, 99% or more identical thereto; or a sequence capable ofhybridizing under stringent conditions described herein (e.g., highlystringent conditions) to a nucleotide sequence encoding the amino acidsequence as shown in FIG. 2A (SEQ ID NO:21), FIG. 5 (e.g., SEQ IDNO:49), or the amino acid sequence of the heavy chain variable region ofthe antibody produced by the NS0 cell line having ATCC Accession NumberPTA-3709 or PTA-4174.

In another embodiment, the isolated nucleic acid comprises a nucleotidesequence encoding at least one, preferably two, and most preferablythree, CDRs of the heavy chain variable region of the anti-PSMA antibodychosen from the amino acid sequences of SEQ ID NO:1, 2, and 3, or 29, 30and 31, or 93, 94, and 95, or 99, 100 and 101, or a CDR sequence whichdiffers by one or two amino acids from the sequences described herein.In yet another embodiment, the isolated nucleic acid comprises anucleotide sequence encoding CDRs 1, 2, or 3 shown in FIG. 4A (SEQ IDNO:23), in SEQ ID NO:51, in FIG. 7B (SEQ ID NO:125), in FIG. 9A (SEQ IDNO:73), or in FIG. 11A (SEQ ID NO:83), or a complement thereof, or asequence encoding a CDR that differs by one or two amino acids from thesequences described herein.

In another embodiment, the isolated nucleic acid comprises a nucleotidesequence encoding at least one, preferably two, three and mostpreferably four amino acid sequences from the heavy chain variableframework region of the anti-PSMA modified antibody chosen from SEQ IDNO:9, 10, 11 and 12, or 37, 38, 39 and 40, or a sequence at least 85%,90%, 95%, 99% or more identical thereto.

In yet another embodiment, the isolated nucleic acid comprises ananti-PSMA modified antibody light chain variable region nucleotidesequence having a sequence as shown in FIG. 4B (SEQ ID NO:25), FIG. 8A(SEQ ID NO:56), or SEQ ID NO:52, or a complement thereof (e.g., SEQ IDNO:26 or 58), or the nucleotide sequence of the light chain variableregion of the antibody produced by the NS0 cell line having ATCCAccession Number PTA-3709 or PTA-4174; a sequence at least 85%, 90%,95%, 99% or more identical thereto; or a sequence capable of hybridizingunder stringent conditions described herein (e.g., highly stringentconditions) to the nucleotide sequence as shown in FIG. 4B (SEQ IDNO:25), FIG. 8A (SEQ ID NO:56), SEQ ID NO:52, or a complement thereof(e.g., SEQ ID NO:26 or 58), or the nucleotide sequence of the lightchain variable region of the antibody produced by the NS0 cell linehaving ATCC Accession Number PTA-3709 or PTA-4174, or a complementthereof. In another embodiment, the isolated nucleic acid encodes ananti-PSMA modified antibody light chain variable region amino acidsequence having a sequence as shown in FIG. 2B (SEQ ID NO:22) or in FIG.6 (e.g., SEQ ID NO:50), the amino acid sequence of the light chainvariable region of the antibody produced by the NS0 cell line havingATCC Accession Number PTA-3709 or PTA-4174; a sequence at least 85%,90%, 95%, 99% or more identity thereto; or a sequence capable ofhybridizing under stringent conditions described herein (e.g., highlystringent conditions) to a nucleotide sequence encoding the amino acidsequence as shown in FIG. 2B (SEQ ID NO:22) or in FIG. 6 (SEQ ID NO:50),or the amino acid sequence of the light chain variable region of theantibody produced by the NS0 cell line having ATCC Accession NumberPTA-3709 or PTA-4174.

In another embodiment, the isolated nucleic acid comprises a nucleotidesequence encoding at least one, preferably two, and most preferablythree, CDRs of the light chain variable region of the anti-PSMA antibodychosen from the amino acid sequences of SEQ ID NO:4, 5, and 6, or 32,33, and 34, or 96, 97, and 98, or 102, 103, and 104, or a sequenceencoding a CDR which differs by one or two amino acids from thesequences described herein.

In yet another embodiment, the isolated nucleic acid comprises anucleotide sequence selected encoding CDRs 1-3 of the light chainvariable nucleotide sequence shown in SEQ ID NO:25, or a sequenceencoding a CDR which differs by one or two amino acids from thesequences described herein. In another embodiment, the isolated nucleicacid comprises a nucleotide sequence encoding at least one, preferablytwo, three and most preferably four amino acid sequences from the lightchain variable framework region of the anti-PSMA modified antibodychosen from SEQ ID NO:13, 14, 15, and 16, or 41, 42, 43, and 44, or asequence at least 85%, 90%, 95%, 99% or more identical thereto.

In a preferred embodiment, there is an isolated first and second nucleicacid which have nucleotide sequences encoding a light chain and theheavy chain variable regions of an anti-PSMA antibody, respectively,wherein each isolated nucleic acid has at least one, two, three, four,five and preferably all CDRs chosen from the amino acid sequences of SEQID NO:1, 2, 3, 4, 5, and 6, or 29, 30, 31, 32, 33 and 34, or 93, 94, 95,96, 97, and 98, or 99, 100, 101, 102, 103, and 104, or sequence encodinga CDR which differs by one or two amino acids from the sequencesdescribed herein.

The nucleic acid can encode only the light chain or the heavy chainvariable region, or can also encode an antibody light or heavy chainconstant region, operatively linked to the corresponding variableregion. In one embodiment, the light chain variable region is linked toa constant region chosen from a kappa or a lambda constant region.Preferably, the light chain constant region is from a lambda type (e.g.,a human type lambda). In another embodiment, the heavy chain variableregion is linked to a heavy chain constant region of an antibody isotypeselected from the group consisting of IgG (e.g., IgG1, IgG2, IgG3,IgG4), IgM, IgA1, IgA2, IgD, and IgE. Preferably, the heavy chainconstant region is from an IgG (e.g., an IgG1) isotype, e.g., a humanIgG1.

Nucleic acids of the invention can be chosen for having codons, whichare preferred, or non-preferred, for a particular expression system.E.g., the nucleic acid can be one in which at least one codon, atpreferably at least 10%, or 20% of the codons has been altered such thatthe sequence is optimized for expression in E. coli, yeast, human,insect, NS0, or CHO cells.

In a preferred embodiment, the nucleic acid differs (e.g., differs bysubstitution, insertion, or deletion) from that of the sequencesprovided, e.g., as follows: by at least one but less than 10, 20, 30, or40 nucleotides; at least one but less than 1%, 5%, 10% or 20% of thenucleotides in the subject nucleic acid. If necessary for this analysisthe sequences should be aligned for maximum homology. “Looped” outsequences from deletions or insertions, or mismatches, are considereddifferences. The differences are, preferably, differences or changes atnucleotides encoding a non-essential residue(s) or a conservativesubstitution(s).

In one embodiment, the first and second nucleic acids are linked, e.g.,contained in the same vector. In other embodiments, the first and secondnucleic acids are unlinked, e.g., contained in different vectors.

In another aspect, the invention features host cells and vectors (e.g.,recombinant expression vectors) containing the nucleic acids, e.g., thefirst and second nucleic acids, of the invention.

Prokaryotic or eukaryotic host cells may be used. The terms “host cell”and “recombinant host cell” are used interchangeably herein. Such termsrefer not only to the particular subject cell, but to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein. A host cell can be any prokaryotic, e.g., bacterial cells suchas E. coli, or eukaryotic, e.g., insect cells, yeast, or preferablymammalian cells (e.g., cultured cell or a cell line). Other suitablehost cells are known to those skilled in the art.

Preferred mammalian host cells for expressing the anti-PSMA antibodies,or antigen-binding fragments thereof, include Chinese Hamster Ovary (CHOcells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980)Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectablemarker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol.Biol. 159:601-621), lymphocytic cell lines, e.g., NS0 myeloma cells andSP2 cells, COS cells, and a cell from a transgenic animal, e.g., e.g.,mammary epithelial cell.

In another aspect, the invention features a vector, e.g., a recombinantexpression vector. The recombinant expression vectors of the inventioncan be designed for expression of the modified antibodies, or anantigen-binding fragment thereof, in prokaryotic or eukaryotic cells.For example, polypeptides of the invention can be expressed in E. coli,insect cells (e.g., using baculovirus expression vectors), yeast cellsor mammalian cells. Suitable host cells are discussed further inGoeddel, (1990) Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to an antibody encoded therein,usually to the constant region of the recombinant antibody.

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that are operativelylinked and control the expression of the antibody chain genes in a hostcell.

In an exemplary system for recombinant expression of a modifiedantibody, or antigen-binding portion thereof, of the invention, arecombinant expression vector encoding both the antibody heavy chain andthe antibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to enhancer/promoter regulatory elements (e.g., derived fromSV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLPpromoter regulatory element or an SV40 enhancer/AdMLP promoterregulatory element) to drive high levels of transcription of the genes.The recombinant expression vector also carries a DHFR gene, which allowsfor selection of CHO cells that have been transfected with the vectorusing methotrexate selection/amplification. The selected transformanthost cells are cultured to allow for expression of the antibody heavyand light chains and intact antibody is recovered from the culturemedium. Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody from theculture medium.

Purification and Conjugation of Anti-PSMA Antibodies

The invention features methods of purifying an anti-PSMA antibody from asample. The method includes: providing a harvested anti-PSMA antibodyproduct; subjecting the harvested product to an antibody bindingchromatography step, and subjecting the anti-PSMA antibody product toone or more ion exchange chromatography steps to thereby obtain purifiedanti-PSMA. The term “purified” anti-PSMA antibody, as used herein,refers to an anti-PSMA antibody product that is substantially free ofcellular material when produced by a cell which expresses the anti-PSMAantibody. The language “substantially free of cellular material”includes preparations of anti-PSMA antibody in which the protein isseparated from cellular components of the cells in which it is produced.In one embodiment, the language “substantially free of cellularmaterial” includes preparations of anti-PSMA antibody having less thanabout 30% (by dry weight) of non-anti-PSMA antibody protein (alsoreferred to herein as a “protein impurity” or “contaminating protein”),more preferably less than about 20% of non-anti-PSMA antibody protein,still more preferably less than about 10% of non-anti-PSMA antibodyprotein, and most preferably less than about 5% non-anti-PSMA antibodyprotein. When the anti-PSMA antibody is obtained (i.e., harvested) fromculture media, it is also preferably substantially free of a componentof the culture medium, i.e., components of the culture medium representless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the dry weight of the proteinpreparation.

The term “harvested anti-PSMA antibody” as used herein refers to ananti-PSMA antibody obtained from culture media or from a cell.

The antibody binding chromatography can be, e.g., a Protein A and/or aProtein G chromatography step. Preferably, the anti-PSMA antibodyproduct is subjected to more than one ion exchange chromatography step.The ion exchange chromatography can be: anion exchange chromatography,cation exchange chromatography or both. In a preferred embodiment, anionexchange chromatography is performed using one or more of: Q SEPHAROSEFAST FLOW®, MACROPREP HIGH Q SUPPORT®, DEAE SEPHAROSE FAST FLOW®, andMACRO-PREP DEAE®. In a preferred embodiment, cation exchangechromatography is performed using one or more of: SP SEPHAROSE FASTFLOW®, SOURCE 30S®, CM SEPHAROSE FAST FLOW®, MACRO-PREP CM SUPPORT®, andMACRO-PREP HIGH S SUPPORT®.

In another aspect, the invention features a method of purifying ananti-PSMA antibody product. The method includes: providing a harvestedanti-PSMA antibody product; subjecting the anti-PSMA antibody product toProtein A chromatography; subjecting the anti-PSMA antibody product toanion exchange chromatography; and subjecting the anti-PSMA antibodyproduct to cation exchange chromatography, to thereby obtain purifiedanti-PSMA antibody. Preferably, anion exchange chromatography isperformed using one or more of: Q SEPHAROSE FAST FLOW®, MACROPREP HIGH QSUPPORT®, DEAE SEPHAROSE FAST FLOW®, and MACRO PREP DEAE®. Preferably,cation exchange chromatography is performed using one or more of: SPSEPHAROSE FAST FLOW®, SOURCE 30S®, CM SEPHAROSE FAST FLOW®, MACRO-PREPCM SUPPORT®, and MACRO-PREP HIGH S SUPPORT®.

An anti-PSMA antibody, e.g., a modified anti-PSMA antibody, orantigen-binding portion thereof, e.g., a purified anti-PSMA antibodydescribed herein, can be derivatived or linked to another molecularentity.

As discussed herein, the molecular entity can be a radiolabel, e.g., aradioisotope, e.g., a radioisotope which is an α-emitter, a β-emitter, aγ-emitter or a β- and γ-emitter. Radioisotopes useful as therapeuticagents include yttrium (⁹⁰Y), lutetium (¹⁷⁷Lu), actinium (²²⁵Ac),praseodymium, astatine (²¹¹At), rhenium (¹⁸⁶Re), bismuth (²¹²Bi or²¹³Bi), and rhodium (¹⁸⁸Rh). Radioisotopes useful as labels, e.g., foruse in diagnostics, include iodine (¹³¹I or ¹²⁵I), indium (¹¹¹In),technetium (⁹⁹mTc), phosphorus (³²P), carbon (¹⁴C), and tritium (³H), orone of the therapeutic isotopes listed above.

The invention provides methods of radio labeling an anti-PSMA antibody,e.g., [a modified anti-PSMA antibody such as those described herein. Themethod includes contacting an anti-PSMA antibody, e.g., an anti-PSMAantibody described herein, with a chelating agent, e.g., 1,4,7,10tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA), and thencontacting the anti-PSMA antibody with a radiolabel, to thereby producea conjugated antibody. In one embodiment, the chelating agent is DOTAand the DOTA is activated in situ prior to conjugation with an anti-PSMAantibody. Prior to contacting the activated DOTA with an anti-PSMAantibody, the quantity of in situ activated DOTA, e.g., DOTA-NHS, in asample can be determined. DOTA can be activated in situ, e.g., usingcoupling reagents such as NHS and EDAC. Thus, in some embodiments, theinvention includes a method of analyzing a sample of DOTA to determinethe quantity of activated DOTA in the sample.

Since hydrolysis of DOTA-NHS in large excess of water is a first orderreaction, based on real time kinetics of DOTA-NHS hydrolysis, thepercentage of DOTA-NHS in a sample can be determined. The method allowsdirect quantification of DOTA-NHS by quantitating DOTA. The method alsoprovides an evaluation of the availability of DOTA-NHS before theconjugation reaction. The availability of DOTA-NHS in different batchescan be determined by quantifying DOTA at different time points duringthe real time kinetics of hydrolysis. The method includes: determiningthe quantity of DOTA in a batch of in situ activated DOTA preferably atseveral time points, e.g., two, three, four, five, or more time pointsduring the hydrolysis of DOTA, to thereby directly evaluate the quantityof DOTA and indirectly of activated DOTA, e.g., DOTA-NHS, in the sample.Thus, the method can include: determining a reference standard for thequantity of DOTA-NHS in a sample activated in situ based upon theconcentration of DOTA in the sample over time during hydrolysis.

Methods of determining a reference standard to quantitate the amount ofDOTA-NHS in a sample are described, e.g., in Example 23. The inventioncan further include methods of conjugating an anti-PSMA antibody with aradiolabel using a DOTA chelating agent which is activated in situ,wherein the amount of DOTA that the anti-PSMA antibody is contacted withis determined based upon a comparison of the quantity of active DOTA,e.g., DOTA-NHS, in the sample of DOTA to be used and a referencestandard for quantitating DOTA. The method includes adjusting theconcentration of active DOTA, e.g., DOTA-NHS, used to contact theanti-PSMA antibody based upon the quantity of active DOTA in the sampleas compared to the reference standard. Preferably, the concentration ofDOTA-NHS used to conjugate the anti-PSMA antibody is an amount thatresults in a ratio of about 2 to 10, preferably 4 to 8, more preferably,5 to 7 DOTA-NHS per anti-PSMA antibody.

The invention also features methods of making multiple batches of a DOTAconjugated anti-PSMA antibody preparation using in situ activated DOTA,wherein average ratio of DOTA-NHS per antibody per batch varies by lessthan 3 DOTA-NHS chelating agents per antibody, preferably less than 2,or 1 DOTA-NHS chelating agents per antibody from batch to batch.Preferably, the average ratio of DOTA-NHS per antibody is about 4 to 8,preferably about 5 to 7 (e.g., 5.5 to 6.5) DOTA-NHS per antibody frombatch to batch. As used herein, “batch” refers to a quantity of anythingproduced at one operation, e.g., a quantity of a compound produced allin one operation. A “batch of drug” is a selection quantity of a drug,e.g., that was produced at one operation, e.g., in a single process.

The invention also features methods of radiolabeling an anti-PSMAantibody, e.g., a modified anti-PSMA antibody such as those describedherein, using a chelating agent which is available in its active form ina substantially pure form. The term “substantially pure” refers to acomposition of an activated chelating agent which contains less than 5%,3%, 2%, 1% other components or contaminants. For example, the chelatingagent can be DOTA which is commercially available as a pure DOTA-NHSmono-active ester from Macrocyclics. In other embodiments, the chelatingagent can be, e.g., a substantially pure DOTA-HOBT active ester, or ap-nitrophenyl active ester. The use of a purified active form of achelating agent such as DOTA can allow for control over the amount ofDOTA-NHS used in the conjugation process, thereby decreasing variabilitybetween batches of DOTA conjugated anti-PSMA antibody preparations. Themethod includes contacting an anti-PSMA antibody, e.g., an anti-PSMAantibody described herein, with a substantially pure composition of achelating agent. In one embodiment, the anti-PSMA antibody is contactedwith an activated chelating agent, e.g., activated DOTA, e.g., DOTA-NHS,at a ratio of 7 to 1, 9 to 1, 11 to 1, 15 to 1, 20 to 1 or 30 to 1DOTA-NHS molecules per antibody. Preferably, the input ration ofactivated DOTA to antibody is 7 to 1, 9 to 1 or 11 to 1.

The invention also features methods of radio labeling an anti-PSMAantibody, e.g., an anti-PSMA antibody described herein, which includescontacting an anti-PSMA antibody with a chelating agent, e.g., activatedDOTA, to form a reaction mixture; removing excess chelating agent, e.g.,unbound DOTA, from the reaction mixture; and contacting the reactionmixture with a radiolabel, to thereby form a radiolabeled anti-PSMAantibody. In a preferred embodiment, the excess chelating agent isremoved such that the reaction mixture includes less than 20%, 15%, 10%,5%, 2%, 1%, or 0.5% excess chelating agent, e.g., unbound DOTA. In otherembodiments, the amount of excess chelating agent present in thereaction mixture is reduced by at least 2-fold, preferably 5- to 10-foldafter the removal step. The removal of excess chelating agent can resultin at least a 10%, 20%, 30%, 40% or more increase in radiolabelefficiency as compared to the percentage of radiolabeled anti-PSMAconjugates obtained without removing the excess chelating agent.

In other aspects, the molecular entity can be a therapeutic agent, e.g.,a cytotoxic moiety, e.g., a therapeutic drug, molecules of plant,fungal, or bacterial origin, or biological proteins (e.g., proteintoxins) or particles (e.g., recombinant viral particles, e.g., via aviral coat protein), or mixtures thereof. For example, the anti-PSMAantibody, or antigen binding fragment thereof, can be coupled to amolecule of plant or bacterial origin (or derivative thereof), e.g., amaytansinoid (e.g., maytansinol or the DM1 maytansinoid, see FIG. 15), ataxane, or a calicheamicin. The invention provides methods ofconjugating an anti-PSMA antibody, e.g., a modified anti-PSMA antibodysuch as those described herein, with a therapeutic drug such as amaytansinoid, e.g., DM1. The method includes contacting an anti-PSMAantibody, e.g., an anti-PSMA antibody described herein, with a linker,e.g., a disulfide linker such as SSP, to form a reaction mixture,contacting the reaction mixture with a therapeutic agent, e.g., amaytansinoid such as DM1, and obtaining a composition which includesanti-PSMA antibody conjugated to the therapeutic agent, e.g., DM1. In apreferred embodiment, the method includes: contacting the anti-PSMAantibody with an amount of linker such that the ratio of linker toantibody in the reaction mixture is about 7:1, 6:1, 5:1, 4:1 or 3:1. Theratio of linker to antibody in the reaction mixture can be selected,e.g., to result in a yield of anti-PSMA antibody in the composition ofat least about 65%, preferably at least 70%, 75%, 80%, 85%, 90%, 95% orgreater. Accordingly, the invention features methods of preparing ananti-PSMA antibody, e.g., an anti-PSMA antibody described herein,conjugated to a therapeutic agent such as DM1 which results obtaining acomposition having a yield of at least about 70% or greater of anti-PSMAantibody, by providing a ratio of linker to antibody of less than 7:1.Preferably, the ratio is about 6:1 to about 4:1.

Pharmaceutical Compositions

In another aspect, the present invention provides compositions, e.g.,pharmaccutically acceptable compositions, which include an antibodymolecule described herein (e.g., a modified antibody molecule asdescribed herein), formulated together with a pharmaceuticallyacceptable carrier.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, isotonic and absorption delaying agents,and the like that are physiologically compatible. The carrier can besuitable for intravenous, intramuscular, subcutaneous, parenteral,rectal, spinal or epidermal administration (e.g., by injection orinfusion).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, liposomes and suppositories. The preferred form dependson the intended mode of administration and therapeutic application.Typical preferred compositions are in the form of injectable orinfusible solutions. The preferred mode of administration is parenteral(e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In apreferred embodiment, the antibody is administered by intravenousinfusion or injection. In another preferred embodiment, the antibody isadministered by intramuscular or subcutaneous injection.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high antibody concentration. Sterileinjectable solutions can be prepared by incorporating the activecompound (i.e., antibody or antibody portion) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The antibodies and antibody-fragments described herein can beadministered by a variety of methods known in the art, although for manytherapeutic applications, the preferred route/mode of administration isintravenous injection or infusion. As described in the Examples below,the anti-PSMA antibody can be administered by intravenous infusion at arate of less than 10 mg/min, preferably less than or equal to 5 mg/minto reach a dose of about 1 to 500 mg/m², preferably about 10 to 400mg/m², about 18 to 350 mg/m², and more preferably, about 250-280 mg/m².The anti-PSMA antibody can be administered in a single dose or inmultiple doses. The dosage schedule can be varied, such that theantibody is administered once, twice, three or more times per week forany number of weeks or the antibody is administered more than once(e.g., two, three, four, five, six, seven times) with administrationoccurring once a week, once every two, three, four, five, six, seven,eight, nine or ten weeks. In a preferred embodiment, the anti-PSMAantibody molecule can be administered once a week for six weeks for atotal of six doses, or twice a week for six weeks for a total of twelvedoses. For example, the anti-PSMA antibody molecule, e.g., an antibodydescribed herein molecule, e.g., an antibody molecule conjugated to atherapeutic agent such as DM1, can be administered in doses of about 13to 23 mg/m² (e.g., 18 mg/m²), 27 to 37 mg/m² (e.g., 32 mg/m²), 46 to 56mg/m² (e.g., 51 mg/m²), or 66 to 76 mg/m² (e.g., 71 mg/m²), twice a weekfor six weeks. As another example, the anti-PSMA antibody molecule,e.g., an antibody molecule described herein, e.g, an antibody moleculeconjugated to a therapeutic agent such as DM1, can be administered indoses of about 66 to 76 mg/m² (e.g., 71 mg/m²), 87 to 97 mg/m² (e.g., 92mg/m²), 115 to 125 mg/m² (e.g., 120 mg/m²), or 151 to 161 mg/m² (e.g.,156 mg/m²), once a week for six weeks. In another example, the anti-PSMAantibody molecule, e.g., an antibody molecule described herein, e.g., anantibody molecule conjugated to a radioisotope, e.g., ¹⁷⁷Lu or ⁹⁰Y, canbe administered in multiple doses with administration of theradioisotope coupled antibody molecule occurring once a week, once everytwo, three, four, five, six, seven, eight, nine or ten weeks. Forantibody molecules of the invention coupled to ¹⁷⁷Lu, multiple doses canbe administered such that each dose is administered at about 65% or lessthan the maximum tolerated dose (MTD) of the antibody coupled to ¹⁷⁷Luand the doses are administered once every week, two weeks, three weeks,four weeks, five weeks, six weeks, seven weeks, eight weeks or more. Forexample, multiple doses of an antibody molecule described herein coupledto ¹⁷⁷Lu can be administered such that each dose is less than 65%, 60%,55%, 50%, 45%, 40%, 35% or less than the MTD of the antibody moleculecoupled to ¹⁷⁷Lu. Multiple doses of ¹⁷⁷Lu conjugated antibodies can beadministered such that each dose is about the same or one or more of thedoses can differ from the others, e.g., one or more of the doses candiffer from the others so long as no dose exceeds 65% of the MTD of theantibody molecule coupled to ¹⁷⁷Lu. As will be appreciated by theskilled artisan, the route and/or mode of administration will varydepending upon the desired results. In certain embodiments, the activecompound may be prepared with a carrier that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, an antibody or antibody portion of the inventionmay be orally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

Therapeutic compositions can be administered with medical devices knownin the art.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion orantibody-conjugate of the invention is about 0.025-125 mg/kg, morepreferably about 1-10 mg/kg. As described in Examples 10 and 12, theanti-PSMA antibody can be administered by intravenous infusion at a rateof less than 10 mg/min, preferably less than or equal to 5 mg/min toreach a dose of about 1 to 500 mg/m², preferably about 10 to 400 mg/m²,about 18 to 350 mg/m², and more preferably, about 250-280 mg/m². It isto be noted that dosage values may vary with the type and severity ofthe condition to be alleviated. It is to be further understood that forany particular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the modifiedantibody or antibody fragment may vary according to factors such as thedisease state, age, sex, and weight of the individual, and the abilityof the antibody or antibody portion to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the modified antibody or antibodyfragment is outweighed by the therapeutically beneficial effects. A“therapeutically effective dosage” preferably inhibits a measurableparameter, e.g., tumor growth rate by at least about 20%, morepreferably by at least about 40%, even more preferably by at least about60%, and still more preferably by at least about 80% relative tountreated subjects. The ability of a compound to inhibit a measurableparameter, e.g., cancer, can be evaluated in an animal model systempredictive of efficacy in human tumors. Alternatively, this property ofa composition can be evaluated by examining the ability of the compoundto inhibit, such inhibition in vitro by assays known to the skilledpractitioner.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Also within the scope of the invention are kits comprising an anti-PSMAantibody described herein, preferably a modified antibody, orantigen-binding fragment thereof. The kit can include one or more otherelements including: instructions for use; other reagents, e.g., a label,a therapeutic agent, or an agent useful for chelating, or otherwisecoupling, an antibody to a label or therapeutic agent, or aradioprotective composition; devices or other materials for preparingthe antibody for administration; pharmaceutically acceptable carriers;and devices or other materials for administration to a subject.Instructions for use can include instructions for diagnosticapplications of the anti-PSMA antibodies (or antigen-binding fragmentthereof) to detect PSMA, in vitro, e.g., in a sample, e.g., a biopsy orcells from a patient having a cancer or prostatic disorder, or in vivo.The instructions can include instructions for therapeutic applicationincluding suggested dosages (e.g., suggested dosages for single ormultiple doses, e.g., as described herein) and/or modes ofadministration, e.g., in a patient with a cancer or prostatic disorder.Other instructions can include instructions on coupling of the antibodyto a chelator, a label or a therapeutic agent, or for purification of aconjugated antibody, e.g., from unreacted conjugation components. Asdiscussed above, the kit can include a label, e.g., any of the labelsdescribed herein. As discussed above, the kit can include a therapeuticagent, e.g., a therapeutic agent described herein. The kit can include areagent useful for chelating or otherwise coupling a label ortherapeutic agent to the antibody, e.g., a reagent discussed herein. Forexample, a macrocyclic chelating agent, preferably1,4,7,10-tetraazacyclododecane-N, N′,N″,N′″-tetraacetic acid (DOTA), canbe included. The DOTA can be supplied as a separate component or theDOTA (or other chelator or conjugating agent) can be supplied alreadycoupled to the antibody. Additional coupling agents, e.g., an agent suchas N-hydroxysuccinimide (NHS), can be supplied for coupling thechelator, e.g., DOTA, to the antibody. As another example, the kit cancontain a linker for conjugating the antibody to a therapeutic agent,e.g., a disulfide linker, e.g., SPP. The linker can be supplied as aseparate component or the linker can be supplied already coupled to theantibody. In other embodiments, the kit can include one or more reagentsuseful for linking the antibody to a therapeutic agent. For example, thekit can include an antibody which has been modified, e.g., activated,such that it includes a moiety which allows linkage to a therapeuticagent. The kit can also include a therapeutic agent for conjugating tothe antibody, such as TAXOL® (paclitaxel), cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicine, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycinD,1-dihydrotestosterone, glucocorticoids, procaine, tetracaine,lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (seeU.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092,5,585,499, 5,846,545) and/or analogs or homologs thereof. In someapplications the antibody will be reacted with other components, e.g., achelator or a label or therapeutic agent, e.g., a radioisotope, e.g.,yttrium or lutetium. In such cases the kit can include one or more of areaction vessel to carry out the reaction or a separation device, e.g.,a chromatographic column, for use in separating the finished productfrom starting materials or reaction intermediates. In some embodiments,the kit can include instructions for making the conjugated antibody,instructions for evaluating the conjugated antibody, e.g., instructionsfor determining the amount of conjugated antibody.

The kit can further contain at least one additional reagent, such as adiagnostic or therapeutic agent, e.g., a diagnostic or therapeutic agentas described herein, and/or one or more additional anti-PSMA antibodies(or fragments thereof), formulated as appropriate, in one or moreseparate pharmaceutical preparations.

The kit can further contain a radioprotectant. The radiolytic nature ofisotopes, e.g., ⁹⁰Yttrium (⁹⁰Y) is known. In order to overcome thisradiolysis, radioprotectants may be included, e.g., in the reactionbuffer, as long as such radioprotectants are benign, meaning that theydo not inhibit or otherwise adversely affect the labeling reaction,e.g., of an isotope, such as of ⁹⁰Y, to the antibody.

The formulation buffer of the present invention may include aradioprotectant such as human serum albumin (HSA) or ascorbate, whichminimize radiolysis due to yttrium or other strong radionuclides. Otherradioprotectants are known in the art and can also be used in theformulation buffer of the present invention, i.e., free radicalscavengers (phenol, sulfites, glutathione, cysteine, gentisic acid,nicotinic acid, ascorbyl palmitate, H0P(:0)H₂I glycerol, sodiumformaldehyde sulfoxylate, Na₂S₂0., Na₂S₂0₃, and S0₂, etc.).

A preferred kit is one useful for radiolabeling a chelator-conjugatedprotein or peptide with a therapeutic radioisotope for administration toa patient. The kit includes (i) a vial containing chelator-conjugatedantibody, (ii) a vial containing formulation buffer for stabilizing andadministering the radiolabeled antibody to a patient, and (iii)instructions for performing the radiolabeling procedure. The kitprovides for exposing a chelator-conjugated antibody to the radioisotopeor a salt thereof for a sufficient amount of time under amiableconditions, e.g., as recommended in the instructions. A radiolabeledantibody having sufficient purity, specific activity and bindingspecificity is produced. The radiolabeled antibody may be diluted to anappropriate concentration, e.g., in formulation buffer, and administereddirectly to the patient with or without further purification. Thechelator-conjugated antibody may be supplied in lyophilized form.

A further preferred kit is one that includes an anti-PSMA antibodydescribed herein conjugated to a therapeutic agent, e.g., TAXOL®(paclitaxel), cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D,1-dihydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids,e.g., maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat.Nos. 5,475,092, 5,585,499, 5,846,545) and/or analogs or homologsthereof. In a more preferred embodiment, the kit includes an anti-PSMAantibody described herein conjugated to DM1, e.g., deJ591-DM1, andinstructions for use, e.g., instructions for therapeutic applicationincluding suggested dosages and/or modes of administration, e.g., in apatient with a cancer or prostatic disorder.

Uses of the Invention

The antibodies of the invention (e.g., the modified antibodies describedherein) have in vitro and in vivo diagnostic, therapeutic andprophylactic utilities. For example, these antibodies can beadministered to cells in culture, e.g. in vitro or ex vivo, or in asubject, e.g., in vivo, to treat, prevent, and/or diagnose a variety ofdisorders, such as cancers (prostatic and non-prostatic cancers), aswell as non-cancerous prostatic conditions (e.g., benign hyperplasticprostatic disorders).

As used herein, the term “subject” is intended to include human andnon-human animals. Preferred human animals include a human patienthaving a disorder characterized by abnormal functioning of aPSMA-expressing cell, e.g., a cancer cell or a prostatic cell. The term“non-human animals” of the invention includes all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dog, cow,chickens, amphibians, reptiles, etc.

In one embodiment, the subject is a human subject. Alternatively, thesubject can be a mammal expressing a PSMA-like antigen with which anantibody of the invention cross-reacts. An antibody molecule of theinvention can be administered to a human subject for therapeuticpurposes (discussed further below). Moreover, an anti-PSMA antibody (orfragment thereof) can be administered to a non-human mammal expressingthe PSMA-like antigen with which the modified antibody cross-reacts(e.g., a primate, pig or mouse) for veterinary purposes or as an animalmodel of human disease. Regarding the latter, such animal models may beuseful for evaluating the therapeutic efficacy of antibodies of theinvention (e.g., testing of dosages and time courses of administration).

Therapeutic Uses

In one embodiment, the invention provides a method of treating, e.g.,ablating or killing, a cell, e.g., a prostatic cell (e.g., a cancerousor non-cancerous prostatic cell, e.g., a normal, benign or hyperplasticprostatic epithelial cell), or a malignant, non-prostatic cell, e.g.,cell found in a non-prostatic solid tumor, a soft tissue tumor, or ametastatic lesion (e.g., a cell found in renal, urothelial (e.g.,bladder), testicular, colon, rectal, lung (e.g., non-small cell lungcarcinoma), breast, liver, neural (e.g., neuroendocrine), glial (e.g.,glioblastoma), pancreatic (e.g., pancreatic duct) cancer and/ormetastasis, melanoma (e.g., malignant melanoma), or soft tissuesarcoma). Methods of the invention include the steps of contacting thecell, or a nearby cell, e.g., a vascular endothelial cell proximate tothe cell, with an anti-PSMA antibody, e.g., a modified anti-PSMAantibody, e.g., a modified antibody as described herein, in an amountsufficient to treat, e.g., ablate or kill, the cell.

The subject method can be used on cells in culture, e.g. in vitro or exvivo. For example, prostatic cells (e.g., malignant or normal, benign orhyperplastic prostate epithelial cells) or non-prostatic cancerous ormetastatic cells (e.g., renal, an urothelial, colon, rectal, lung,breast or liver, cancerous or metastatic cells) can be cultured in vitroin culture medium and the contacting step can be effected by adding theanti-PSMA antibody or fragment thereof, to the culture medium. Themethod can be performed on cells (e.g., prostatic cells, ornon-prostatic cancerous or metastatic cells) present in a subject, aspart of an in vivo (e.g., therapeutic or prophylactic) protocol. For invivo embodiments, the contacting step is effected in a subject andincludes administering the anti-PSMA antibody or fragment thereof to thesubject under conditions effective to permit both binding of theantibody or fragment to the cell, or the vascular endothelial cellproximate to the cell, and the treating, e.g., the killing or ablatingof the cell.

Examples of prostatic disorders that can be treated or preventedinclude, but are not limited to, genitourinary inflammation (e.g.,inflammation of smooth muscle cells) as in prostatitis; benignenlargement, for example, nodular hyperplasia (benign prostatichypertrophy or hyperplasia); and cancer, e.g., adenocarcinoma orcarcinoma, of the prostate and/or testicular tumors. “Recurrence” or“recurrent” prostate cancer, as used herein, refers to an increase inPSA levels after an anti-cancer treatment (e.g., prostatectomy orradiation) to greater than 0.4 ng/dL in two consecutive tests spaced bya one month period. After an anti-cancer treatment such as aprostatectomy or radiation, PSA levels drop to low and in some casesundetectable levels in the blood. This drop in PSA levels below 0.4ng/dL allows PSA levels to be followed in order to determine if therehas been cancer recurrence in a subject. Cancer recurrence can occurover a short period of time from the anti-cancer treatment, e.g., a fewmonths after treatment, or can occur several years after an anti-cancertreatment. For example, in prostate cancer patients, recurrence canhappen several years after an anti-cancer treatment, e.g., up to 4, 5,6, 7, 8, 9, 10, 12, 14, 15 years after treatment. Recurrence can beclassified as “local recurrence” or “distant recurrence”. “Localrecurrence” refers to cancers which recur in tissue or organs adjacentto or proximate to the cancerous tissue or organ. For example, insubjects having prostate cancer, local recurrence can occur in tissuenext to the prostate, in the seminal vesicles, the surrounding lymphnodes in the pelvis, the muscles next to the prostate, and the rectumand/or walls of the pelvis. “Distant recurrence” refers to cancers whichrecur distant from the cancerous tissue or organ. For example, insubjects having prostate cancer, distant recurrence includes cancerswhich spread to the bones or other organs.

As used herein, the term “cancer” is meant to include all types ofcancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness.

Examples of non-prostatic cancerous disorders include, but are notlimited to, solid tumors, soft tissue tumors, and metastatic lesions.Examples of solid tumors include malignancies, e.g., sarcomas,adenocarcinomas, and carcinomas, of the various organ systems, such asthose affecting lung, breast, lymphoid, gastrointestinal (e.g., colon),and genitourinary tract (e.g., renal, urothelial cells), pharynx.Adenocarcinomas include malignancies such as most colon cancers, rectalcancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma ofthe lung, cancer of the small intestine and cancer of the esophagus.Metastatic lesions of the aforementioned cancers can also be treated orprevented using the methods and compositions of the invention.

The subject method can be useful in treating malignancies of the variousorgan systems, such as those affecting lung, breast, lymphoid,gastrointestinal (e.g., colon), bladder, genitourinary tract (e.g.,prostate), pharynx, as well as adenocarcinomas which includemalignancies such as most colon cancers, renal-cell carcinoma, prostatecancer and/or testicular tumors, non-small cell carcinoma of the lung,cancer of the small intestine and cancer of the esophagus.

In other embodiments, the antibodies of the invention can be used forthe diagnosis and treatment of a subject experiencing pain or sufferingfrom a pain-associated disorder. Preferably, the subject is a human,e.g., a patient with pain or a pain-associated disorder disclosedherein. For example, the subject could have a disease of the prostate,e.g., benign prostatic hyperplasia or prostate cancer, or non-prostatecancer, e.g., a cancer having vasculature which expresses PSMA (e.g.,renal, urothelial (e.g., bladder), testicular, colon, rectal, lung(e.g., non-small cell lung carcinoma), breast, liver, neural (e.g.,neuroendocrine), glial (e.g., glioblastoma), or pancreatic (e.g.,pancreatic duct) cancer, melanoma (e.g., malignant melanoma), or softtissue sarcoma). The pain can be associated with bones, as well as withobstructive voiding symptoms due to enlarged prostate, e.g., urinaryhesitancy or diminished urinary stream, frequency or nocturia. Thetreatment of pain using the anti-PSMA antibodies of the invention canlead to a decreased or dramatically lowered need, or even eliminate theneed, for analgesics, e.g., narcotics. In addition, by reducing pain,the methods of treatment can restore the mobility of, e.g., limbs, thathave become dysfunctional as a result of pain associated with movement.

Methods of administering antibody molecules are described above.Suitable dosages of the molecules used will depend on the age and weightof the subject and the particular drug used. The antibody moleculesdescribed herein can be used as competitive agents for ligand binding toinhibit, reduce an undesirable interaction.

In one embodiment, the anti-PSMA antibodies, e.g., the modifiedanti-PSMA antibodies, or antigen-binding fragments thereof, can be usedto kill or ablate cancerous cells and normal, benign hyperplastic, andcancerous prostate epithelial cells in vivo. For example, the anti-PSMAantibodies can be used to treat or prevent a disorder described herein.The antibodies, e.g., the modified antibodies, (or fragments thereof)can be used by themselves or conjugated to a second agent, e.g., acytotoxic drug, radioisotope, or a protein, e.g., a protein toxin or aviral protein. This method includes: administering the antibody, aloneor conjugated to a cytotoxic drug, to a subject requiring suchtreatment.

Since the anti-PSMA antibodies (or fragments thereof) recognize normal,benign hyperplastic, and cancerous prostate epithelial cells, any suchcells to which the antibodies bind are destroyed. Although suchadministration may destroy normal prostate epithelial cells, this is notproblematic, because the prostate is not required for life or survival.Although the prostate may indirectly contribute to fertility, this isnot likely to be a practical consideration in patients receiving thetreatment of the present invention. In the case of cancerous tissues,since the antibodies recognize vascular endothelial cells that areproximate to cancerous cells, binding of the antibody/cytotoxic drugcomplex to these vascular endothelial cells destroys them, therebycutting off the blood flow to the proximate cancerous cells and, thus,killing or ablating these cancerous cells. Alternatively, theantibodies, by virtue of their binding to vascular endothelial cellsthat are proximate to cancerous cells, are localized proximate to thecancerous cells. Thus, by use of suitable antibodies (including thosecontaining substances effective to kill cells nondiscriminatingly butonly over a short range), cells in cancerous tissue (including cancerouscells) can be selectively killed or ablated.

The antibodies of the present invention may be used to deliver a varietyof therapeutic agents, e.g., a cytotoxic moiety, e.g., a therapeuticdrug, a radioisotope, molecules of plant, fungal, or bacterial origin,or biological proteins (e.g., protein toxins) or particles (e.g., arecombinant viral particles, e.g., via a viral coat protein), ormixtures thereof. The therapeutic agent can be an intracellularly activedrug or other agent, such as short-range radiation emitters, including,for example, short-range, high-energy α-emitters, as described herein.In some preferred embodiments, the anti-PSMA antibody, or antigenbinding fragment thereof, can be coupled to a molecule of plant orbacterial origin (or derivative thereof), e.g., a maytansinoid (e.g.,maytansinol or the DM1 maytansinoid, see FIG. 15). DM1 is asulfhydryl-containing derivative of maytansine that can be linked toantibodies via a linker, e.g., a disulfide linker that releases DM1 wheninside target cells. Maytansine is a cytotoxic agent that effects cellkilling by preventing the formation of microtubules and depolymerizationof extant microtubules. It is 100- to 1000-fold more cytotoxic thananticancer agents such as doxorubicin, methotrexate, and vinca alkyloid,which are currently in clinical use. Alternatively, the anti-PSMAantibody, or antigen binding fragment thereof, can be coupled to ataxane, a calicheamicin, a proteosome inhibitor, or a topoisomeraseinhibitor.[(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(3-mercaptoacetyl)amino]propyl]amino]butyl]Boronic acid is a suitable proteosome inhibitor.N,N′-bis[2-(9-methylphenazine-1-carboxamido)ethyl]-1,2-ethanediamine isa suitable topoisomerase inhibitor. Enzymatically active toxins andfragments thereof are exemplified by diphtheria toxin A fragment,nonbinding active fragments of diphtheria toxin, exotoxin A (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,α-sacrin, certain Aleurites fordii proteins, certain Dianthin proteins,Phytolacca americana proteins (PAP, PAPII and PAP-S), Morodica charantiainhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin,mitogillin, restrictocin, phenomycin, and enomycin. In a preferredembodiment, the anti-PSMA antibody is conjugated to maytansinoids, e.g.,maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos.5,475,092, 5,585,499, 5,846,545). Procedures for preparing enzymaticallyactive polypeptides of the immunotoxins are described in WO84/03508 andWO85/03508, which are hereby incorporated by reference, and in theappended Examples below. Examples of cytotoxic moieties that can beconjugated to the antibodies include adriamycin, chlorambucil,daunomycin, methotrexate, neocarzinostatin, and platinum.

To kill or ablate normal, benign hyperplastic, and cancerous prostateepithelial cells, a first antibody, e.g., a modified antibody, can beconjugated with a prodrug which is activated only when in closeproximity with a prodrug activator. The prodrug activator is conjugatedwith a second antibody, e.g., a second modified antibody according tothe present invention, preferably one that binds to a non-competing siteon the prostate specific membrane antigen molecule. Whether two modifiedantibodies bind to competing or non-competing binding sites can bedetermined by conventional competitive binding assays. For example,monoclonal antibodies J591, J533, and E99 bind to competing bindingsites on the prostate specific membrane antigen molecule. Monoclonalantibody J415, on the other hand, binds to a binding site that isnon-competing with the site to which J591, J533, and E99 bind. Thus, forexample, the first modified antibody can be one of J591, J533, and E99,and the second modified antibody can be J415. Alternatively, the firstmodified antibody can be J415, and the second modified antibody can beone of J591, J533, and E99. Drug-prodrug pairs suitable for use in thepractice of the present invention are described in Blakely et al.,“ZD2767, an Improved System for Antibody-directed Enzyme Prodrug TherapyThat Results in Tumor Regressions in Colorectal Tumor Xenografts,”(1996) Cancer Research, 56:3287-3292, which is hereby incorporated byreference.

A number of linkers can be used to couple the therapeutic agent to theanti-PSMA antibody. For example, a disulfide linkage can be used, asdescribed below in Example 15, and in Saito et al., Adv. Drug DeliveryReviews, 55:199-215 (2003); inter al/a. Linkers that are sensitive tothe lower pH found in endosomes can also be used, including hydrazones,ketals and/or aconitic acids. A hybrid linker can also be used, e.g., alinker with two or more potential cleavage sites, e.g., a disulfide anda hydrazone. Peptidase-sensitive linkers can also be used, e.g.,tumor-specific peptidases, for example, linkers sensitive to cleavage byPSA. PEG linkers can also be used (Wüest et al., Oncogene 21:4257-4265(2002)). Exemplary linkers include hydrazone and disulfide hybridlinkers (Seattle Genetics; see Hamann et al, Bioconjugate Chem. 13:47-58(2002); Hamann et al., Bioconjug Chem. 13(1):40-6 (2002)); SPP(Immunogen); and a variety of linkers available from PierceBiotechnology, Inc. In some embodiments, the linker is SSP (a disulfidelinker, available from Immunogen), and the ratio of linker to antibodycan be varied from, e.g., 7:1 (7 linkers per antibody molecule) to 4:1.Preferably, the ratio is less than 7:1, e.g., 6.3:1.

Alternatively, the antibody, e.g., the modified antibody, can be coupledto high energy radiation emitters, for example, a radioisotope, such as¹³¹I, a γ-emitter, which, when localized at the tumor site, results in akilling of several cell diameters. See, e.g., S. E. Order, “Analysis,Results, and Future Prospective of the Therapeutic Use of RadiolabeledAntibody in Cancer Therapy”, Monoclonal Antibodies for Cancer Detectionand Therapy, R. W. Baldwin et al. (eds.), pp 303-316 (Academic Press1985), which is hereby incorporated by reference. Other suitableradioisotopes include α-emitters, such as ²¹²Bi, ²¹³Bi, and ²¹¹At, andβ-emitters, such as ¹⁸⁶Re and ⁹⁰Y. Radiotherapy is expected to beparticularly effective, because prostate epithelial cells and vascularendothelial cells within cancers are relatively radiosensitive.Moreover, Lu¹¹⁷ may also be used as both an imaging and cytotoxic agent.

Radioimmunotherapy (RIT) using antibodies labeled with ¹³¹I, ⁹⁰Y, and¹⁷⁷Lu is under intense clinical investigation. There are significantdifferences in the physical characteristics of these three nuclides andas a result, the choice of radionuclide can be important in order todeliver maximum radiation dose to the tumor. The higher beta energyparticles of ⁹⁰Y may be good for bulky tumors, but it may not benecessary for small tumors and especially bone metastases, (e.g. thosecommon to prostate cancer). The relatively low energy beta particles of¹³¹I are ideal, but in vivo dehalogenation of radioiodinated moleculesis a major disadvantage for internalizing antibody. In contrast, ¹⁷⁷Luhas low energy beta particle with only 0.2-0.3 mm range and deliversmuch lower radiation dose to bone marrow compared to ⁹⁰Y. In addition,due to longer physical half-life (compared to ⁹⁰Y), the tumor residencetimes are higher. As a result, higher activities (more mCi amounts) of¹⁷⁷Lu labeled agents can be administered with comparatively lessradiation dose to marrow. There have been several clinical studiesinvestigating the use of ¹⁷⁷Lu labeled antibodies in the treatment ofvarious cancers. (Mulligan T et al. (1995) Clin Cancer Res. 1:1447-1454; Meredith R F, et al. (1996) J Nucl Med 37:1491-1496; AlvarezR D, et al. (1997) Gynecologic Oncology 65: 94-101).

The antibodies of the invention can also be conjugated or fused to viralsurface proteins present on viral particles. For example, a single-chainanti-PSMA antibody of the present invention could be fused (e.g., toform a fusion protein) to a viral surface protein. Alternatively, awhole anti-PSMA antibody of the present invention, or a fragmentthereof, could be chemically conjugated (e.g., via a chemical linker) toa viral surface protein. Preferably, the virus is one that fuses withendocytic membranes, e.g., an influenza virus, such that the virus isinternalized along with the anti-PSMA antibody and thereby infectsPSMA-expressing cells. The virus can be genetically engineered as acellular toxin. For example, the vims could express or induce theexpression of genes that are toxic to cells, e.g., cell death promotinggenes. Preferably, such viruses would be incapable of viral replication.

The antibodies, e.g., the modified antibodies of the invention, can beused directly in vivo to eliminate antigen-expressing cells via naturalcomplement or antibody-dependent cellular cytotoxicity (ADCC). Modifiedantibody molecules of the invention, which have complement bindingsites, such as portions from IgG1, -2, or -3 or IgM which bindcomplement can also be used in the presence of complement. In oneembodiment, ex vivo treatment of a population of cells comprising targetcells with a binding agent of the invention and appropriate effectorcells can be supplemented by the addition of complement or serumcontaining complement. Phagocytosis of target cells coated with modifiedantibodies or fragments thereof of the invention can be improved bybinding of complement proteins. In another embodiment, target cellscoated with the modified antibodies or fragments thereof can also belysed by complement.

The antibodies, e.g., the modified antibodies, of the present inventioncan be used and sold together with equipment, as a kit, to detect theparticular label.

Also encompassed by the present invention is a method of killing orablating cells which involves using the antibodies described herein,e.g., the modified antibodies for preventing a PSMA-related disorder.For example, these materials can be used to prevent or delay developmentor progression of prostate or other cancers.

Use of the therapeutic methods of the present invention to treatprostate and other cancers has a number of benefits. Since theantibodies according to the present invention only target cancerouscells (such as cells of cancerous tissues containing vascularendothelial cells) and prostate epithelial cells, other tissue isspared. As a result, treatment with such antibodies is safer,particularly for elderly patients. Treatment according to the presentinvention is expected to be particularly effective, because it directshigh levels of antibodies, e.g., modified antibodies, such as antibodiesor binding portions thereof, probes, or ligands, to the bone marrow andlymph nodes where prostate cancer metastases and metastases of manyother cancers predominate. Moreover, the methods of the presentinvention are particularly well-suited for treating prostate cancer,because tumor sites for prostate cancer tend to be small in size and,therefore, easily destroyed by cytotoxic agents. Treatment in accordancewith the present invention can be effectively monitored with clinicalparameters, such as, in the case of prostate cancer, serum prostatespecific antigen and/or pathological features of a patient's cancer,including stage, Gleason score, extracapsular, seminal, vesicle orperineural invasion, positive margins, involved lymph nodes, diseaserelated pain, e.g., bone pain, etc. Alternatively, these parameters canbe used to indicate when such treatment should be employed.

The invention also features methods of treating pain, e.g., reducingpain, experienced by a subject having or diagnosed with prostatedisease, e.g., benign prostatic hyperplasia or prostate cancer, ornon-prostate cancer, e.g., a cancer having vasculature which expressesPSMA (e.g., renal, urothelial (e.g., bladder), testicular, colon,rectal, lung (e.g., non-small cell lung carcinoma), breast, liver,neural (e.g., neuroendocrine), glial (e.g., glioblastoma), or pancreatic(e.g., pancreatic duct) cancer, melanoma (e.g., malignant melanoma), orsoft tissue sarcoma). The methods include administering an anti-PSMAantibody as described herein, e.g., a modified anti-PSMA antibody, to asubject in an amount sufficient to treat, e.g., reduce, the painassociated with prostate disease or non-prostate cancer. The subject mayhave no signs of prostate disease or non-prostate cancer other than,e.g., elevated levels of serum PSA and the sensation of pain. Patientsthat have prostate cancer often experience bone pain, as well as, painassociated with obstructive voiding symptoms due to enlarged prostate,e.g., urinary hesitancy or diminished urinary stream, frequency ornocturia. The treatment of pain using the anti-PSMA antibodies of theinvention can lead to a decreased or dramatically lowered need, or eveneliminate the need, for analgesics, e.g., narcotics. By reducing pain,the methods of treatment can restore the mobility of, e.g., limbs, thathave become dysfunctional as a result of pain associated with movement.

Because the antibodies, e.g., the modified antibodies, of the presentinvention bind to living prostate cells, therapeutic methods fortreating prostate cancer using these antibodies are not dependent on thepresence of lysed prostate cells. For the same reasons, diagnostic andimaging methods which determine the location of living normal, benignhyperplastic, or cancerous prostate epithelial cells (as well asvascular endothelial cells within cancers) are much improved byemploying the antibodies of the present invention. In addition, theability to differentiate between living and dead prostate cells can beadvantageous, especially to monitor the effectiveness of a particulartreatment regimen.

The antibodies, e.g., the modified antibodies, or antigen-bindingportions thereof, of the present invention bind to extracellular domainsof prostate specific membrane antigens or portions thereof in normal,benign hyperplastic, and cancerous prostate epithelial cells as well asvascular endothelial cells proximate to cancerous cells. As a result,when practicing the methods of the present invention to kill, ablate, ordetect normal, benign hyperplastic, and cancerous prostate epithelialcells as well as vascular endothelial cells proximate to cancerouscells, the antibodies, e.g., the modified antibodies, bind to all suchcells, not only to cells which are fixed or cells whose intracellularantigenic domains are otherwise exposed to the extracellularenvironment. Consequently, binding of the antibodies, e.g., the modifiedantibodies, is concentrated in areas where there are prostate epithelialcells, irrespective of whether these cells are fixed or unfixed, viableor necrotic. Additionally or alternatively, these antibodies, e.g.,these modified antibodies, or binding portions thereof, bind to and areinternalized with prostate specific membrane antigens or portionsthereof in normal, benign hyperplastic, and cancerous prostateepithelial cells.

Combination Therapy

The anti-PSMA antibodies described herein, e.g., the modified anti-PSMAantibodies, or antigen-binding fragments thereof, may be used incombination with other therapies. Administered “in combination”, as usedherein, means that two (or more) different treatments are delivered tothe subject during the course of the subject's affliction with thedisorder, e.g., the two or more treatments are delivered after thesubject has been diagnosed with the disorder and before the disorder hasbeen cured or eliminated or treatment has ceased for other reasons. Insome embodiments, the delivery of one treatment is still occurring whenthe delivery of the second begins, so that there is overlap in terms ofadministration. This is sometimes referred to herein as “simultaneous”or “concurrent delivery.” In other embodiments, the delivery of onetreatment ends before the delivery of the other treatment begins. Insome embodiments of either case, the treatment is more effective becauseof combined administration. For example, the second treatment is moreeffective, e.g., an equivalent effect is seen with less of the secondtreatment, or the second treatment reduces symptoms to a greater extent,than would be seen if the second treatment were administered in theabsence of the first treatment, or the analogous situation is seen withthe first treatment. In some embodiments, delivery is such that thereduction in a symptom, or other parameter related to the disorder isgreater than what would be observed with one treatment delivered in theabsence of the other. The effect of the two treatments can be partiallyadditive, wholly additive, or greater than additive. The delivery can besuch that an effect of the first treatment delivered is still detectablewhen the second is delivered.

Exemplary therapeutic agents include TAXOL® (paclitaxel), cytochalasinB, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicine, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D,1-dihydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g.,maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos.5,475,092, 5,585,499, 5,846,545) and analogs or homologs thereof.Therapeutic agents include, but are not limited to, antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, TAXOL® (paclitaxel) and maytansinoids).In particular, vinblastine, estramustine, and/or mitoxantrone can beused in combination with the DM1-coupled anti-PSMA antibodies describedherein. In another embodiment, the anti-PSMA antibody is coupled to atherapeutic agent other than DM1, and the antibody is administered incombination with a taxane, e.g., paclitaxel or TAXOL® (paclitaxel).

In other embodiments, the anti-PSMA antibodies are administered incombination with other therapeutic treatment modalities, includingsurgery, radiation, cryosurgery, and/or thermotherapy. Such combinationtherapies may advantageously utilize lower dosages of the administeredtherapeutic agents, thus avoiding possible toxicities or complicationsassociated with the various monotherapies.

In other embodiments, the anti-PSMA antibodies are administered incombination with another antigen-specific antibody, e.g., an antibodythat is conjugated to a therapeutic agent, e.g., an antibody thattargets an antigen other than PSMA, e.g., an antigen on a non-PSMAexpressing cell. The method can further include administering theanti-PSMA antibody with two, three, four or more antigen-specificantibodies. The anti-PSMA antibody and additional antigen-specificantibodies can be conjugated, e.g., with the same or differenttherapeutic agents or labels, or one or more of the antibodies can beunconjugated.

Anti-PSMA antibodies of the invention can be administered in combinationwith one or more of the existing modalities for treating prostatecancers, including, but not limited to: surgery (e.g., radicalprostatectomy); radiation therapy (e.g., external-beam therapy whichinvolves three dimensional, conformal radiation therapy where the fieldof radiation is designed to conform to the volume of tissue treated;interstitial-radiation therapy where seeds of radioactive compounds areimplanted using ultrasound guidance; and a combination of external-beamtherapy and interstitial-radiation therapy); hormonal therapy, which canbe administered before or following radical prostatectomy or radiation(e.g., treatments which reduce serum testosterone concentrations, orinhibit testosterone activity, e.g., administering a leuteinizinghormone-releasing hormone (LHRH) analog or agonist (e.g., Lupron,Zoladex, leuprolide, buserelin, or goserelin) or antagonists (e.g.,Abarelix). Non-steroidal anti-androgens, e.g., flutamide, bicalutimade,or nilutamide, can also be used in hormonal therapy, as well assteroidal anti-androgens (e.g., cyproterone acetate or megastrolacetate), estrogens (e.g., diethylstilbestrol), PROSCAR™, secondary ortertiary hormonal manipulations (e.g., involving corticosteroids (e.g.,hydrocortisone, prednisone, or dexamethasone), ketoconazole, and/oraminogluthethimide), inhibitors of 5a-reductase (e.g., finisteride),herbal preparations (e.g., PC-SPES), hypophysectomy, and adrenalectomy.Furthermore, hormonal therapy can be performed intermittently or usingcombinations of any of the above treatments, e.g., combined use ofleuprolide and flutamide.

In other embodiments, the anti-PSMA antibodies, e.g., the modifiedanti-PSMA antibodies, are administered in combination with animmunomodulatory agent, e.g., IL-1, 24, 6, or 12, or interferon alpha orgamma. As described in Example 14 below, the combination of antibodieshaving a human constant regions and IL-2 potentially is expected toenhance the efficacy of the monoclonal antibody. IL-2 will function toaugment the reticuloendothelial system to recognize antigen-antibodycomplexes by its effects on NK cells and macrophages. Thus, bystimulating NK cells to release IFN, GM-CSF, and TNF, these cytokineswill increase the cell surface density of Fc receptors, as well as thephagocytic capacities of these cells. Therefore, the effector arm ofboth the humoral and cellular arms will be artificially enhanced. Thenet effect will be to improve the efficiency of monoclonal antibodytherapy, so that a maximal response may be obtained. A small number ofclinical trials have combined IL-2 with a monoclonal antibody (Albertiniet al. (1997) Clin Cancer Res 3:1277-1288; Frost et al. (1997) Cancer80:317-333; Kossman et al. (1999) Clin Cancer Res 5:2748-2755). IL-2 canbe administered by either bolus or continuous infusion. Accordingly, theantibodies of the invention can be administered in combination with IL-2to maximize their therapeutic potential.

The combination therapy can also include a composition of the presentinvention coformulated with, and/or coadministered with, one or moreadditional therapeutic agents, e.g., one or more anti-cancer agents,cytotoxic or cytostatic agents, hormone treatment, vaccines, and/orother immunotherapies.

Diagnostic Uses

In one aspect, the present invention provides a diagnostic method fordetecting the presence of a PSMA protein in vitro (e.g., in a biologicalsample, such as a tissue biopsy, e.g., from a cancerous or prostatictissue) or in vivo (e.g., in vivo imaging in a subject). The methodincludes: (i) contacting the sample with an anti-PSMA antibody orfragment thereof (e.g., a modified anti-PSMA antibody), or administeringto the subject, the anti-PSMA antibody (e.g., the modified anti-PSMAantibody); (optionally) (ii) contacting a reference sample, e.g., acontrol sample (e.g., a control biological sample, such as plasma,tissue, biopsy) or a control subject)); and (iii) detecting formation ofa complex between the anti-PSMA antibody, and the sample or subject, orthe control sample or subject, wherein a change, e.g., a statisticallysignificant change, in the formation of the complex in the sample orsubject relative to the control sample or subject is indicative of thepresence of PSMA in the sample.

Preferably, the anti-PSMA antibody (or fragment thereof) is directly orindirectly labeled with a detectable substance to facilitate detectionof the bound or unbound antibody. Suitable detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials and radioactive materials, as described above and described inmore detail below.

Complex formation between the anti-PSMA antibody and PSMA can bedetected by measuring or visualizing either the antibody (or antibodyfragment) bound to the PSMA antigen or unbound antibody (or antibodyfragment). Conventional detection assays can be used, e.g., anenzyme-linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) ortissue immunohistochemistry. Alternative to labeling the anti-PSMAantibody, the presence of PSMA can be assayed in a sample by acompetition immunoassay utilizing standards labeled with a detectablesubstance and an unlabeled anti-PSMA antibody. In this assay, thebiological sample, the labeled standards and the PSMA binding agent arecombined and the amount of labeled standard bound to the unlabeledantibody is determined. The amount of PSMA in the sample is inverselyproportional to the amount of labeled standard bound to the PSMA bindingagent.

In still another embodiment, the invention provides a method fordetecting the presence of PSMA-expressing cancerous tissues(particularly the vascular endothelial cells therein) and normal, benignhyperplastic, and cancerous prostate epithelial cells in vivo. Themethod includes (i) administering to a subject (e.g., a patient having acancer or prostatic disorder) an anti-PSMA antibody, preferably amodified antibody, conjugated to a detectable marker; (ii) exposing thesubject to a means for detecting said detectable marker to thePSMA-expressing tissues or cells. Particularly preferred antibodiesinclude modified antibodies having CDRs from any of a J591, J415, J533or E99, and in particular deimmunized versions of these antibodies,particularly deJ591 or deJ415.

Examples of labels useful for diagnostic imaging in accordance with thepresent invention are radiolabels such as ¹³¹I, ¹¹¹In, ¹²³I, ^(99m)Tc,³²P, ¹²⁵I, ³H, ¹⁴C, and ¹⁸⁸Rh, fluorescent labels such as fluoresceinand rhodamine, nuclear magnetic resonance active labels, positronemitting isotopes detectable by a positron emission tomography (“PET”)scanner, chemiluminescers such as luciferin, and enzymatic markers suchas peroxidase or phosphatase. Short-range radiation emitters, such asisotopes detectable by short-range detector probes, such as atransrectal probe, can also be employed. These isotopes and transrectaldetector probes, when used in combination, are especially useful indetecting prostatic fossa recurrences and pelvic nodal disease. Themodified antibody can be labeled with such reagents using techniquesknown in the art. For example, see Wensel and Meares (1983)Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York, which ishereby incorporated by reference, for techniques relating to theradiolabeling of antibodies. See also, D. Colcher et al. (1986) Meth.Enzymol. 121: 802-816, which is hereby incorporated by reference.

In the case of a radiolabeled antibody, the antibody is administered tothe patient, is localized to the tumor bearing the antigen with whichthe antibody reacts, and is detected or “imaged” in vivo using knowntechniques such as radionuclear scanning using e.g., a gamma camera oremission tomography. See e.g., A. R. Bradwell et al., “Developments inAntibody Imaging”, Monoclonal Antibodies for Cancer Detection andTherapy, R. W. Baldwin et al., (eds.), pp 65-85 (Academic Press 1985),which is hereby incorporated by reference. Alternatively, a positronemission transaxial tomography scanner, such as designated Pet VIlocated at Brookhaven National Laboratory, can be used where theradiolabel emits positrons (e.g., ¹¹C, ¹⁸F, ¹⁵O, and ¹³N).

Fluorophore and chromophore labeled antibodies, e.g., modifiedantibodies, can be prepared from standard moieties known in the art.Since antibodies and other proteins absorb light having wavelengths upto about 310 nn, the fluorescent moieties should be selected to havesubstantial absorption at wavelengths above 310 nm and preferably above400 nm. A variety of suitable fluorescent compounds and chromophores aredescribed by Stryer (1968) Science, 162:526 and Brand, L. et al. (1972)Annual Review of Biochemistry, 41:843-868, which are hereby incorporatedby reference. The antibodies can be labeled with fluorescent chromophoregroups by conventional procedures such as those disclosed in U.S. Pat.Nos. 3,940,475, 4,289,747, and 4,376,110, which are hereby incorporatedby reference.

One group of fluorescers having a number of the desirable propertiesdescribed above is the xanthene dyes, which include the fluoresceinsderived from 3,6-dihydroxy-9-henylxanthhydrol and resamines andrhodamines derived from 3,6-diamino-9-phenylxanthydrol and lissanimerhodamine B. The rhodamine and fluorescein derivatives of9-o-carboxyphenylxanthhydrol have a 9-o-carboxyphenyl group. Fluoresceincompounds having reactive coupling groups such as amino andisothiocyanate groups such as fluorescein isothiocyanate andfluorescamine are readily available. Another group of fluorescentcompounds are the naphthylamines, having an amino group in the α or βposition.

In cases where it is important to distinguish between regions containinglive and dead prostate epithelial cells or to distinguish between liveand dead prostate epithelial cells, the antibodies of the presentinvention (or other modified antibodies of the present invention),labeled as described above, can be coadministered along with an antibodyor other modified antibody which recognizes only living or only deadprostate epithelial cells labeled with a label which can bedistinguished from the label used to label the subject antibody. Bymonitoring the concentration of the two labels at various locations ortimes, spatial and temporal concentration variations of living and deadnormal, benign hyperplastic, and cancerous prostate epithelial cells canbe ascertained. In particular, this method can be carried out using thelabeled antibodies of the present invention, which recognize both livingand dead epithelial prostate cells, and labeled 7E11 antibodies(Horoszewicz et al. (1987) Anticancer Research 7:927-936), whichrecognize only dead epithelial prostate cells.

In other embodiments, the invention provide methods for determining thedose, e.g., radiation dose, that different tissues are exposed to when asubject, e.g., a human subject, is administered an anti-PSMA antibodythat is conjugated to a radioactive isotope. The method includes: (i)administering an anti-PSMA antibody as described herein, e.g., amodified anti-PSMA antibody, that is labeled with a radioactive isotopeto a subject; (ii) measuring the amount of radioactive isotope locatedin different tissues, e.g., prostate, liver, kidney, or blood, atvarious time points until some or all of the radioactive isotope hasbeen eliminated from the body of the subject; and (iii) calculating thetotal dose of radiation received by each tissue analyzed. Themeasurements can be taken at scheduled time points, e.g., day 1, 2, 3,5, 7, and 12, following administration (at day 0) of the radioactivelylabeled anti-PSMA antibody to the subject. The concentration ofradioisotope present in a given tissue, integrated over time, andmultiplied by the specific activity of the radioisotope can be used tocalculate the dose that a given tissue receives. Pharmacologicalinformation generated using anti-PSMA antibodies labeled with oneradioactive isotope, e.g., a gamma-emitter, e.g., ¹¹¹In, can be used tocalculate the expected dose that the same tissue would receive from adifferent radioactive isotope which cannot be easily measured, e.g., abeta-emitter, e.g., ⁹⁰Y.

Pharmacogenomics

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp.Pharmacol. Physiol. 23:983-985 and Under, M. W. et al. (1997) Clin.Chem. 43:254-266. Differences in metabolism of therapeutics can lead tosevere toxicity or therapeutic failure by altering the relation betweendose and blood concentration of the pharmacologically active drug. Ingeneral, two types of pharmacogenetic conditions can be differentiated.Genetic conditions transmitted as a single factor altering the way drugsact on the body (altered drug action) or genetic conditions transmittedas single factors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. Morespecifically, the term refers the study of how a patient's genesdetermine his or her response to a drug (e.g., a patient's “drugresponse phenotype,” or “drug response genotype.”) Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment according to that individual'sdrug response genotype.

Information generated from pharmacogenomic research can be used todetermine appropriate dosage and treatment regimens for prophylactic ortherapeutic treatment of an individual. This knowledge, when applied todosing or drug selection, can avoid adverse reactions or therapeuticfailure and thus enhance therapeutic or prophylactic efficiency whenadministering a therapeutic composition, e.g., a composition consistingof one or more anti-PSMA antibodies, or derivatized form(s) thereof, toa patient, as a means of treating a disorder, e.g., a cancer orprostatic disorder as described herein.

In one embodiment, a physician or clinician may consider applyingknowledge obtained in relevant pharmacogenomics studies when determiningwhether to administer a pharmaceutical composition, e.g., a compositionconsisting of one or more anti-PSMA antibodies, derivatized form(s)thereof, and optionally a second agent, to a subject. In anotherembodiment, a physician or clinician may consider applying suchknowledge when determining the dosage, e.g., amount per treatment orfrequency of treatments, of a pharmaceutical composition, e.g., apharmaceutical composition as described herein, administered to apatient.

In yet another embodiment, a physician or clinician may determine thegenotypes, at one or more genetic loci, of a group of subjectsparticipating in a clinical trial, wherein the subjects display adisorder, e.g., a cancer or prostatic disorder as described herein, andthe clinical trial is designed to test the efficacy of a pharmaceuticalcomposition, e.g., a composition consisting of one or more anti-PSMAantibodies, and optionally a second agent, and wherein the physician orclinician attempts to correlate the genotypes of the subjects with theirresponse to the pharmaceutical composition.

Deposits

Hybridomas E99, J415, J533, and J591 have been deposited pursuant to,and in satisfaction of, the requirements of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure with the American Type Culture Collection(“A.T.C.C.”) at 10801 University Boulevard, Manassas, Va. 20110-2209.Hybridoma E99 was deposited on May 2, 1996, and received A.T.C.C.Designation Number HB-12101. Hybridoma J415 was deposited on May 30,1996, and received A.T.C.C. Designation Number HB-12109. Hybridomas J533and J591 were deposited on Jun. 6, 1996, and received A.T.C.C.Designation Numbers HB-12127 and HB-12126, respectively.

An NS0 cell line producing deimmunized J591 was deposited with AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209, on Sep. 18, 2001 and assigned Accession Number PTA-3709.An NS0 cell line producing deimmunized J415 was deposited with AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209, on Mar. 21, 2002 and assigned Accession Number PTA-4174.These deposits will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

The following invention is further illustrated by the followingexamples, which should not be construed as further limiting. Thecontents of all references, pending patent applications and publishedpatents, cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1 Chelation of Anti-PSMA Antibodies to ¹¹¹Indium,⁹⁰Yttrium, and ¹⁷⁷Lutetium

The modified anti-PSMA monoclonal antibodies can be radiolabeled with¹¹¹Indium, ⁹⁰Yttrium, or ¹⁷⁷Lutetium by directly coupling one of thefour carboxylic acid groups of the chelator1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) toprimary amines present on the surface of the antibodies. The DOTAconjugated antibody is then purified, sterile filtered, and vialed.Prior to use, the purified antibodies can be mixed with the desiredradiolabel which binds to DOTA.

Chelation Process

Monoclonal antibody deJ591 was conjugated with1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) andsubsequently radiolabeled with ¹¹¹In, ⁹⁰Y and ¹⁷⁷Lu. Radiolabeling andquality control tests were performed on three separate vials of clinicalgrade mAb deJ591.

All reagents used in the conjugation and purification of deJ591 weremade from pyrogen-free water. In the specific case of NH₄OAC buffer andsodium phosphate buffer, the solutions were purified with Chelex 100(Bio-Rad, CA) to remove any metal ions.

Conjugation of Antibody with1,4,7,10-Tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA)

The monoclonal antibody deJ591 was modified with1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) asfollows. Briefly, 25 mg of deJ591 was concentrated in a 30 kDa microsepcentrifugal concentrator (Pall Filtron, MA) and washed with 5×4 ml of 1%DTPA (pH 5.0), over a period of 24 hours. The antibody buffer was thenchanged to 0.1 M phosphate (pH 7.0) using the same centrifugaltechnique. An active ester of DOTA was created by dissolving 146 mg DOTA(0.361 mmoles) and 36 mg N-hydroxysuccinimide (0.313 mmoles) in 2 ml ofwater and adjusting the pH to 7.3 with NaOH, prior to the addition of 10mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (see below).

This reaction mixture was cooled on ice for 1 hour before being added tothe deJ591 solution. The resultant DOTA-deJ591 was separated from theexcess DOTA and other reactants by repeated washing with 0.3 M NH₄OAc(20×4 mL) and centrifugal concentration. The purified conjugate was thensterilized by filtration through a 0.22 μm filter and stored in asterile polypropylene vial at 4° C.

The concentration of the DOTA-deJ591 conjugate was assayed bydetermining the UV absorption at 280 nm and two 50 μL aliquots mixedwith either 20 or 30 μL of a 1.30 mM solution of InCl₃ (0.01 M HCl)spiked with a tracer amount of ¹¹¹In. The mixture is incubated at 37° C.for 16 hours and then analyzed by ITLC, using silica gel impregnatedglass fiber 10 cm strip (ITLC-SG, Gelman, prod. #61885) and an eluant of1% DTPA (pH 6.0). The antibody bound activity remains at the origin andfree ¹¹¹In moves with the solvent front as an ¹¹¹In-DTPA complex. Therelative amounts of ¹¹¹In and ¹¹¹In -DOTA-J591 is determined by cuttingthe ITLC strip at a Rf of 0.5 and counting the two halves with a Na(Tl)Idetector. The number of binding sites is calculated by considering themolar reaction ratio between ¹¹¹In and DOTA-deJ591 and the observedratio of ¹¹¹In and ¹¹¹In-DOTA-J591 detected. Typically, 5.1 molecules ofDOTA are conjugated to deJ591. Table 10 shows the results from twoconjugations of deJ591.

TABLE 10 Calculation of the Mean Number of DOTA Molecules Conjugated todeJ591 Known Observed Mean number of ¹¹¹In/DOTA-J591 ¹¹¹In/DOTA-J591DOTA mols per Test number Reaction ratio TLC ratio mAb A 11.76 1.3385.03 B 17.64 2.469 5.09

Radiolabeling

The following radiolabeling procedure is described for ¹¹¹In, but may beused with other radiolabels such as ⁹⁰Y or ¹⁷⁷Lu. Radiolabeling wasachieved by adding the ¹¹¹In (in dilute HCl) to the ammonium acetatebuffered DOTA-deJ591. To avoid the effects of autoradiolysis on theantibody, the reaction time was minimized and the reaction mixturepurified with a size exclusion column prior to administration. Briefly,a mixture composed of 20 μL of ¹¹¹InCl₃ (8 mCi, 0.01 M HCl, 400 μLDOTA-deJ591 (4 mg/ml, 0.3 M NH₄OAc, pH 7) was allowed to react at 37° C.for 20 minutes. The reaction mixture was then separated on a 16 mLBiogel-P6DG column (Bio-Rad, CA) equilibrated with 4×10 mL of sterile 1%HSA in PBS (USA meets specification for US licensed albumin;manufactured by Central Laboratory Blood Transfusion Service Swiss RedCross, Bern, Switzerland, License No. 647). Once the reaction mixturewas loaded onto the column, it was washed with a further 2 mL of 1% HSAPBS, before the main ¹¹¹In-DOTA-deJ591 fraction was eluted with 5 mL of1% HSA PBS. The purified ¹¹¹In-DOTA-deJ591 was then sterile filteredinto a sterile evacuated vial. Using this method, specific activity of7.6 mCi ¹¹¹In/mg DOTA-deJ591 was achieved.

Alternative Radiolabeling Procedure for ¹¹¹In

The following radiolabeling procedure can be used for the routinepreparation of ¹¹¹In-DOTA-J591 for clinical studies and stabilitystudies. Radiolabeling is achieved by the addition of ¹¹¹In chloride andAmmonium acetate buffer (1 M) to DOTA-J591 solution (8 mg/ml, 0.3 MAmmonium acetate, pH 7). To avoid the effects of autoradiolysis on theantibody, the reaction time has been minimized. The labeled¹¹¹In-DOTA-J591 is purified using a size exclusion column and sterilefiltered using a 0.2 m Millipore membrane filter prior to administrationto patients.

Briefly, ammonium acetate, (10 μL for each mCi of ¹¹¹In) is added to areaction vial containing chloride solution. Subsequently, the DOTA-J591solution (30 mL or 0.24 mg for each mCi of ¹¹¹In) is added to thereaction vial and the mixture is gently mixed and incubated at 37° C.for 20-30 min. An aliquot of the mixture is tested to determine labelingefficiency using ITLC (SG and 5 mM DTP A, pH 5). If the binding isoptimal (>70%), the reaction is stopped by the addition of 10-40 mL of 5mM DTPA.

In order to separate or purify ¹¹¹In-DOTA-J591 from free ¹¹¹In, thereaction mixture is applied on a Biogel-P6DG column (Bio-Rad, CA),prewashed with 4×10 ml of PBS containing 1% Human Serum Albumin (meetsspecification for US licensed albumin; manufactured by CentralLaboratory Blood Transfusion Service Swiss Red Cross, Bern, Switzerland,License No. 647). The ¹¹¹In-DOTA-J591 is eluted from the column usingPBS with 1% HSA and the fractions containing the labeled antibody(typically 5-8 ml) are collected into a sterile container. Followingdetermination of radiochemical purity using ITLC (as before), and if thelabeling efficiency is >95%, the labeled complex is filtered into asterile vial using 0.2 m Filter. The final specific activity istypically 3-5 mCi/mg of antibody.

Radiolabeling with ⁹⁰Y

The procedure is identical to the procedure described above for ¹¹¹In,except the incubation time is 10-15 min. Radiochemical purity of90Y-DOTA-J591 must be >97%.

Radiolabeling with ¹⁷⁷Lu

The procedure is similar to the procedure described above except for twochanges. The amount of Ammonium acetate added is reduced (3-5 mL foreach mCi of ¹⁷⁷Lu) and the incubation time is only 5 min. Radiochemicalpurity of ¹⁷⁷Lu-DOTA-J591 should be >97%.

Radiochemical Purity

The amount of free ¹¹¹In in radiolabeled DOTA-deJ591 preparations wasevaluated using the instant thin layer chromatography method with asilica gel impregnated glass fiber support and a mobile phase of 1% DTPA(pH 5.5). Briefly, a portion of the radiolabeled DOTA-deJ591 was spottedon a 10 cm ITLC-SG strip (Gelman, prod. #61885) and developed in 1% DTPA(pH 5.5). Once the solvent front had reached the end of the strip, itwas removed from the solvent and cut at a Rf of 0.5. The two portionswere assayed for radioactivity and the radiochemical purity determinedusing the following equation:

Radiochemical purity=(Activity in between R _(r) 0 and 0.5)/(Totalactivity in strip)

Immunoreactivity

The immunoreactivity of the ¹¹¹In-DOTA-deJ591 preparations was assessedaccording to the method of Lindmo (Lindmo T. et al. (1994) J. Immunol.Methods, 72:77-89, 1994) that extrapolates the binding of theradiolabeled antibody at an infinite excess of antigen. Briefly, fivetest solutions were prepared (in duplicate) containing 10,000 cpm of¹¹¹In-DOTA-deT591 and various amounts of LNCaP cells, in a total testvolume of 250 μL of 0.2% BSA 10 mM HEPES. The solutions were incubatedat 4° C. for 60 minutes prior to being isolated (by centrifugation) andwashed with ice cold PBS. The membranes were then counted in a gammacounter with standards representing the total radioactivity added. Thedata was plotted using the Lindmo method as the reciprocal of thesubstrate concentration (x-axis) against the reciprocal of the fractionbound (y-axis). The data was then fitted according to a least squareslinear regression method (Sigma Plot) and the y intercept taken as thereciprocal of the immunoreactivity. A similar method using membranesderived from LNCaP cells, and subsequent centrifugation isolation of themembranes, gave similar results. The results gave an averageimmunoreactivity of 72% (see Table 11).

Immunohistochemistry

Immunohistochemistry was performed on the DOTA conjugated, partiallypurified, bulk intermediate deJ591. The results showed that thepreparation was specific to prostate tissue and the reactivity wasequivalent to the naked deJ591 antibody.

Sterility

The sterility of ¹¹¹In-DOTA-deJ591 preparations was determined usingthioglycollate medium according to the method of USP 24/NF 19. Briefly,quadruplicate 0.1 mL samples of the ¹¹¹In-DOTA-deJ591 preparations weretransferred to 15 mL of fluid thioglycollate medium and the mixtureincubated at 35° C. for 14 days. The media were visually inspected onthe 4th, 7th and 14 days of any signs of growth. All three preparationsshowed no growth (See Table 11).

Endotoxin

The endotoxin of ¹¹¹In-DOTA-deJ591 preparations was determined using theLimulus amoebocyte lysate assay according to the USP 24/NF 19. Briefly,a Limulus amoebocyte lysate kit (Bio Whittaker lot #7L3790, sensitivity0.125 EU/mL) was reconstituted with 0.25 mL of test sample. Thequadruplicate test samples, artificially positive test samples, negativecontrols and positive controls were incubated at 37° C. for 60 minutes.Positive results were typified by the formation of a viscous gel thatwas unaffected by 180° inversion. The single preparation gave a value ofless than 5 EU/mL. This assay can (and will) be repeated on the patientdose immediately prior to administration.

TABLE 11 Analytical Results of Radiolabeled ¹¹¹In-DOTA-deJ591 TestResult Radiolabeling yield 85% Radiochemical Purity >99% Immunoreactivity 72% Endotoxin <5 Eu/mL Sterility Sterile

Lot# of deJ591: BIOV983.2-2

Large-Scale Manufacture/Process

The large-scale manufacture of the DOTA conjugated deJ591 antibody isdescribed in the following paragraphs. The major differences from theabove methodology were the use of a stirred cell, instead of a microsepcentrifugal concentrator to concentrate and diafilter the antibody andthe use of a Sephadex G-25 column to remove the unreacted DOTA and otherreagents from the DOTA conjugated antibody. These changes werenecessitated by the increase in scale. The ratios of the startingmaterials are given in Table 12 for a nominal 1000 milligram scale. Theprocess may be scaled up using equivalent ratios of starting materials.

TABLE 12 Unit Ratios of Starting Materials Starting Material Unit RatiodeJ591 antibody   X mgs 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″- 1.25X mgs tetraacetic acid (DOTA) N-hydroxysuccinimide (NHS) 0.275 X mgs 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)  0.3 X mgs

Aseptic practices were observed in order to minimize contamination andenvironmental monitoring was conducted at periodic intervals during themanufacture. All solutions, buffers and reagents used in the conjugationand purification of DOTA-deJ591 antibody were made with Water ForInjection (WFI). Throughout the process, metal free components were usedin the manufacture in order to avoid the chelation of any free metalresidues by the DOTA moiety. In the specific case of ammonium acetatebuffer and sodium phosphate buffer, the solutions were purified withChelex 100 to remove any metal ions. Sterile, pyrogen free and metalfree containers were used to mix reactants. The final bulk sterilefiltration was conducted in an area that meets Class 100 specifications.

The de.T591 was prepared by buffer exchanging the antibody into metalfree, 0.1 M Sodium Phosphate, pH 7.1, over a Chelex 100 (BioRad orequivalent) column. The antibody was then concentrated to approximately10 mg/mL using a Stirred Cell Unit (Millipore or equivalent) equippedwith a 30 kD cut-off membrane. The concentrated antibody was thensterile-filtered through a 0.22 μm filter.

To conjugate one gram of antibody, the active ester of DOTA was preparedby adding 6.3 mL of 0.49 M DOTA in metal free, Sodium Phosphate Buffer,pH 7.1, to 2.7 mL of 0.87 M N-hydroxysuccinimide in metal free, SodiumPhosphate Buffer, pH 7.1. To this mixture, 0.1 N Sodium Hydroxide wasadded until the DOTA was completely dissolved (approximately a 1:1 ratioof 0.1 M Sodium Hydroxide to DOTA/NHS solution). The pH was between 6.9and 7.2. The solution was cooled for not less than 30 minutes at 2-8° C.To the DOTA/NHS solution, 1.5 mL of 1.0 M of EDC in Sodium PhosphateBuffer, pH 7.1, was added and allowed to cool at 2-8° C. for not lessthan 1 hour.

The active DOTA ester was added to 1 gram of antibody and incubatedovernight (12-14 hrs) at 2-8° C. The DOTA conjugated antibody waspurified over a Sephadex G-25 column (Pharmacia or equivalent) in metalfree, 0.3 M Ammonium Acetate Buffer, pH 7.2. The eluate fractioncontaining the DOTA conjugated antibody was concentrated using a StirredCell equipped with a 30 kD cut-off membrane to approximately 10 mg/mL.The DOTA conjugated deJ591 Antibody was then diafiltered in 0.3 MAmmonium Acetate, pH 7.2 to remove any excess reagents and diluted to afinal concentration of 8.0 mg/mL prior to sterile filtering through a0.22 μm filter.

DOTA conjugated deJ591 was tested for concentration, immunoreactivity,conjugation, endotoxin, and sterility. The endotoxin limit is based onthe low clinical dose of the radiolabeled DOTA conjugated deJ591antibody required, which ranges from 1 to 5 mg. Bioburden testing wasperformed on the bulk purified DOTA conjugated antibody instead ofsterility because of the small batch sizes. Sterility (21 CFR 610) willbe performed on the final vialed drug product. The target forimmunoreactivity and number of DOTA moles per antibody was based onprevious clinical experience. DOTA conjugated antibody withimmunoreactivity values of as low as 72% have been successfully used inthe clinic. The number of DOTA moles per antibody is based on theresults from previous clinical lots.

Protein Concentration

A sample of DOTA-deJ591 was analyzed by optical density in aspectrophotometer at a wavelength of 280 nm. The extinction coefficientused for these calculations was A₂₈₀, E_(1cm) ^(0.1%)=1.4. The testsample was suitably diluted to give an absorbance reading in the workingrange of the assay (0.2 OD units to 1.2 OD units, linear, CV less than2%). The acceptable limit for protein concentration is 8.0 mg/mL±0.5mg/mL.

Endotoxin

Samples of DOTA-deJ591 were tested for pyrogens using a validatedLimulus Amebocyte Lysate test (LAL) Gel Clot Assay (BioWhittaker orequivalent). A 0.06 EU/mL sensitivity Lysate was utilized and sampleswere diluted either 1:10 or 1:25 in Endotoxin free water for analysis inorder to overcome the inhibition level of certain chemicals to the gelclot assay. Duplicate determinations were made for each buffer orintermediate sample during processing and the sample values needed to beequal to or less than the value obtained at the dilution level set forthat buffer. A positive and negative control, as well as an inhibitioncontrol, was run with every sample. The proposed acceptable limits werenot more than 5 EU per mg of DOTA-deJ591.

Bioburden

Aliquots of DOTA-deJ591 were directly inoculated in fluid thioglycollateand soybean-casein broth. The media were examined after fourteen days ofincubation. As necessary, both media showed no growth after fourteendays.

Immunoreactivity

The immunoreactivity of the DOTA-deJ591 preparations was assessedaccording to the method of Lindmo (Lindmo T. et al. (1994) J. Immunol.Methods 72:77-89) which extrapolates the binding of the radiolabeledantibody at an infinite amount of excess antigen. Briefly five testsolutions were prepared (in duplicate) containing 10,000 cpm of¹¹¹Indium labeled-DOTA-deJ591 and various amounts of LNCaP cells or cellmembranes, in a total test volume of 250 μL of 0.2% BSA 10 mM HEPES. Thesolutions were incubated at 4° C. for 60 minutes prior to being isolated(by centrifugation) and washed with ice cold PBS. The membranes werethen counted in a gamma counter with standards representing the totalradioactivity added. The data was plotted using the Lindmo method as thereciprocal of the substrate concentration (x-axis) against thereciprocal of the fraction bound (y-axis). The data was then fittedaccording to a least squares linear regression method (Sigma Plot) andthe y intercept used as the reciprocal of the immunoreactivity. Thetarget for immunoreactivity was not less than 75%.

Number of POTA Moles Per Antibody

The number of DOTA bound per antibody was determined using a saturationbinding method with natural occurring isotope of Indium and ¹¹¹Indium.Multiple aliquots (minimum two) of DOTA-deJ591 were mixed with variousamounts, ranging from 10 to 30 μL of a 3.0 mM solution of InCl₃ (0.01 MHCl) spiked with a tracer amount of ¹¹¹In. The mixture was incubated at37° C. for 16 hours and then analyzed by ITLC, using silica gelimpregnated glass fiber 10 cm strip (ITLC-SG, Gelman, or equivalent) andan eluant of 1% DTPA (pH 6.0). The antibody bound activity remains atthe origin and free ¹¹¹In moves with the solvent front as an ¹¹¹In-DTPAcomplex. The relative amounts of ¹¹¹In and ¹¹¹In-DOTA-J591 wasdetermined by cutting the ITLC strip at a R_(f) of 0.5 and counting thetwo halves with a Na(Tl)I detector. The number of binding sites wascalculated by considering the molar reaction ratio between ¹¹¹In andDOTA-deJ591 and the observed ratio of ¹¹¹In and ¹¹¹In-DOTA-J591detected. The target number of DOTA molecules per antibody was between 4and 6.

The analytical results for a sample lot of DOTA conjugated deJ591antibody are shown below in Table 13.

TABLE 13 Test Proposed Acceptable Limits Results Appearance ClearColorless Solution Clear Colorless Solution Concentration 8.0 mg/mL ±0.5 mg/mL 8.4 mg/mL Endotoxin NMT 5 EU per mg <1.2 EU/mg Bioburden Nogrowth No growth Immunoreactivity For Information Only 95% (Target NLT75%) Number of DOTA moles For Information Only 6 per Antibody (Target4-6 DOTA per Antibody)

The DOTA conjugation numbers for a previous lot of DOTA conjugatedantibody (Biov983.2-2) and current Lot 243101 are shown in Table 14. Theaverage number of DOTA moles per antibody for Lot Biov983.2-2 was 5.06and for Lot 243101 was 5.96. Although the number of moles of DOTAconjugated per antibody was slightly higher for Lot 243101, theimmunoreactivity was not affected as shown in Table 15. In fact, theimmunoreactivity for Lot 243101 was higher than that for the comparisonlot, which is beneficial. It should be noted that other small-scaleclinical lots have had immunoreactivity values of greater than 90% (datanot shown).

TABLE 14 Comparison of the Mean Number of DOTA Molecules Conjugated todeJ591 antibody Known ¹¹¹In/DOTA- Observed Mean number deJ591¹¹¹In/DOTA-deJ591 of DOTA Lot number Reaction ratio TLC ratio mols permAb BIOV983.2-2 A 11.76 1.338 5.03 B 17.64 2.469 5.09 Ave 5.06 Lot243101 A 10.98 0.8608 5.90 B 16.46 1.7301 6.03 C 21.95 2.8226 5.74 D32.93 4.3498 6.15 Ave 5.96 A = 10 μL of In-natural/¹¹¹In solution, B =15 μL of In-natural/¹¹¹In solution, C = 20 μL of In-natural/¹¹¹Insolution, D = 30 μL of In-natural/¹¹¹In solution

TABLE 15 Comparison of Immunoreactivity of DOTA-deJ591 Test LotBIOV983.2-2 Lot 243101 Immunoreactivity 72% 95%

Alternatives

An alternative synthesis of the DOTA-J591 immunoconjugate is as follows:956.5 mg of deT591 was diafiltered six times. The antibody wasconcentrated in a 30 kDa microsep centrifugal concentrator(Pall-Filtron, MA) to approximately 15 mg/mL and diluted 12.5 fold withmetal free 0.1 M Sodium phosphate at pH 7.1. This procedure wasperformed six times. An active ester of DOTA was created by mixing 598mg of DOTA (1.48 mmoles) in 5.95 mL 0.1 M metal free phosphate bufferand 132 mg N-hydroxysuccinimide (1.15 mmoles) in 2.7 ml of 0.1 M metalfree phosphate buffer. The pH was adjusted to 6.9-7.2 with NaOH, priorto the addition of 144 mg (0.75 mmoles) of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in 1.45 mL 0.1 M metalfree phosphate buffer. This reaction mixture was filtered through a 0.2micron sterile filter and cooled on ice for 1 hour before being added tothe deJ591 solution and incubated overnight at 2-8° C. for 14-18 hours.The resultant DOTA-deJ591 was separated from the excess DOTA and otherreactants by purifying it through a G-25 column equilibrated in 0.3 Mmetal free ammonium acetate. The purified conjugate was concentrated to10 mg/mL in a stirred cell unit and washed with 0.3 M ammonium acetate,then sterilized by filtration through a 0.22 um filter and stored in asterile polypropylene vial at 2-8° C.

Yet another alternative is to use a conjugation process that involvespure DOTA-NHS mono-active ester (commercially available fromMacrocyclics) so as to achieve better control over the amount and purityof the DOTA-NHS mono-active ester used in the conjugation process, aswell as to limit unanticipated chemical side-reactions produced whenDOTA is activated in-situ. Problems associated with the use of in-situactivated DOTA (i.e., DOTA activated without any purification prior tobeing added to the antibody) include: fluctuation of DOTA/antibodyincorporation ratio due to the variable generation of DOTA-NHS; the useof a large excess of DOTA which needs to be purified away from theconjugated antibody and can compete for binding to radioactive isotopes;unreacted EDAC can result in the cross-linking of lysine and glutamic oraspartic acid residues on proteins and consequently the formation ofundesired protein aggregates; and there is the possibility that two ormore active esters are formed on a significant fraction of the activatedDOTA molecules which enables proteins to become crosslinked and usesadditional carboxylatc groups that are needed for metal coordination.

To minimize the loss of antibody binding activity resulting from itsconjugation to DOTA, it is desirable to incorporate about 2-10,preferably about 5-7 DOTA molecules per antibody molecule. UsingDOTA-NHS monoactivated ester, a 3-30 fold excess of DOTA-NHS relative toantibody produces this desired level of DOTA incorporation. Studies arecurrently being conducted using input ratios of DOTA-NHS to antibody of7:1, 9:1, 11:1, 15:1, 20:1, and 30:1.

The protocol is as follows. Stalling materials: J591 antibody, 10.5mg/ml is sodium phosphate buffer (0.1M, pH 7.1), treated with Chelex 100resin (1 ml resin per 10 mg of antibody); 0.3 M ammonium acetate buffer(pH 7.0), treated with Chelex 100 resin (20 ml resin per one literbuffer); DOTA-NHS.PF6 (FW 646.4), Macrocyclics, Dallas, Tex.Experimental procedures: Three polypropylene vials were separatelycharged with 2.0 ml of J591 antibody (10.5 mg/ml, 143 nmol) and chilledon ice over a period of 30 minutes. 3.3 mg of DOTA-NHS were dissolved in0.356 ml of metal-free water (treated with Chelex 100 resin andpre-chilled on ice for 30 minutes) to give a concentration of 14.3 nmolper microliter. To the three antibody solutions were separately added70, 90, and 110 microliters of this freshly prepared DOTA-NHS solution.The reaction mixtures were slowly stirred with magnetic stirring bars atroom temperature over a period of 4 hours, and then diluted with 0.3 Mammonium acetate buffer (pH 7.0, Chelex 100 treated) to 15 ml inCentriCon-30 for buffer exchange. The concentrates (about 1.5 ml) werethen diluted again to 15 ml and concentrated down to 1.5 ml. Theseconcentrates were then filtered through 0.2 micron filters. TheCentriCon tube and filters were washed with a small amount of ammoniumacetate buffer and filtered into final products (total of 2.0 ml each).

Antibodies conjugated to DOTA using the 7:1, 9:1, and 11:1 input ratioof DOTA-NHS to antibody have been analyzed for ⁹⁰Y binding stability andthe formation of protein aggregates. All three conjugates displayed ahigh percentage of stability after 2-3 days of labeling with ¹¹¹In or⁹⁰Y, in the presence of PBS, DTPA chelate challenger, serum andtransferrin. In addition, all three conjugates displayed little or noprocess-related aggregate formation. The remaining three conjugates,produced using the higher DOTA-NHS to antibody input ratios, arecurrently being analyzed.

The conjugation of DOTA to antibodies is not limited to NHS activatedDOTA. The DOTA-HOBT active ester can be used in place of DOTA-NHS, aswell as other activation methods known in the art, such as the use of amixed anhydride of ethyl chloroformate or isobutyl chloroformate,p-nitrophenyl ester.

Example 2 Pharmacokinetics and Biodistribution of ¹³¹I- and¹¹¹In-Labeled deJ591 and Murine J415 in Nude Mice Bearing LNCaP HumanProstate Tumors

In nude mice bearing PSMA-positive human LNCaP tumors, thepharmacokinetics, biodistribution and tumor uptake of monoclonal deJ591and murine J415 antibodies radiolabeled with ¹³¹I or ¹¹¹In was analyzed.Autoradiographic studies were performed to identify intra-tumoraldistribution of radiolabeled MAbs.

deJ591 and J415 were labeled with ¹³¹I using the iodogen method (seeFranker and Speck (1978), Biochem Biophys Res Commun 80:849-57) to aspecific activity of 400 MBq/mg (21, 23). For ¹¹¹In labeling, the J415and deJ591 antibodies were first conjugated with1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) andthen labeled with ¹¹¹in to produce specific activities of 200 MBq/mg.

Prostate carcinoma cell lines LNCaP, DU145 and PC3 (American TypeCulture Collection, Rockville, Md.) were grown in RPMI 1640,supplemented with 10% fetal calf serum, at a temperature of 37° C. in anenvironment containing 5% CO2. Prior to use, the cells were trypsinized,counted and suspended in Matrigel (Collaborative Biomedical Products,Bedford, Mass.). Nu/Nu Balb C mice 8-10 weeks of age were inoculated, inthe right and left flanks, with a suspension of 5×10⁶ LNCaP cells inMatrigel (BD Biosciences, Bedford Mass.). After a period of 14-18 days,tumors (100-300 mg) had developed. The PSMA-negative DU145 and PC3 cellswere implanted in nude mice in an identical manner.

The PSMA-positive and PSMA-negative tumor bearing mice were injected,via the tail vein, with 80 KBq of the iodinated MAb (400 MBq/mg) in 200μL of PBS (pH 7.4, 0.2% BSA). Groups of animals (3-8/group) weresacrificed after 2, 4 or 6 days. The major organs and tumors wererecovered. The tissue samples were weighed and counted, with appropriatestandards in an automatic NaI (Tl) counter. These measured relativeactivity data (cpm) were background corrected and expressed as apercentage of the injected dose per gram (% ID/g). These data were alsofitted with a least squares regression analysis (Microcal Origin,Northampton, Mass.) to determine the biological clearance of the variousagents. J415, J591 and 7E11 were labeled with ¹¹¹In (100MBq/mg). 80 KBqof the ¹¹¹In labeled MAbs were injected in groups of animals. The % ID/gin various organs and tumor tissues was determined at 2-6 days postinjection in a similar way as described for ¹³¹I labeled MAbs.

For the imaging studies, animals were injected with 2 MBq of¹¹¹In-DOTA-J591. On days 1, 2, 3, 4 and 6 post-injection, the mice weresedated with ketamine/xylazine 100 mg/kg/10 mg/kg IP. The mice wereimaged with a gamma camera (Transcam, ADAC Laboratories, Milpitas,Calif.) equipped with a pinhole collimator. The images were acquired ina 256×256 matrix for 1000 seconds using a 20% window of 245 KeVphotopeak of ¹¹¹In.

For several animals (n=20), harvested tumor samples were immediatelycooled in liquid nitrogen and frozen in embedding medium (O.C.T. 4583,Sakura Finetec, Torrance Calif.). Twenty micron sections were cut andthe tumor sections were either fixed with acetone and placed in directcontact with a sheet of photographic film (Biomax, Kodak, Rochester,N.Y.) or stained with hematoxin/eosin prior to exposure of the film.Tumors from control animals were collected and cut into 10 μm sections.These sections were soaked in tris buffer (170 mM, pH 7.4, with 2 mMCaCl₂ and 5 mM KCl) for 15 minutes, washed with Tris buffer (170 mM, pH7.4) and incubated with ¹³¹I-J591 MAb for 1 hour at 4° C. Non-specificbinding was determined in the presence of 100 nM J591 MAb. Thesesections were then washed 3 times with PBS (containing 0.2% BSA) andonce with Tris buffer prior to being fixed with acetone and then exposedto photographic film.

Biodistribution of ¹³¹I Labeled MAbs

In nude mice bearing LNCaP tumors, the biodistribution and tumor uptakeof ¹³¹I labeled deJ591 and J415 was compared to that of ¹³¹I-7E11. At 2days post injection deJ591, J415, and 7E11 had similar tumor uptake andblood pool activity. On day 6, the tumor uptake of J415 (15.4±1.1) wassignificantly higher compared to that of deJ591 (9.58±1.1). The bloodactivity of both deJ591 and J415 was significantly lower compared tothat of 7E11. With the ¹³¹I labeled MAbs, the tumor/blood andtumor/muscle ratios were significantly higher with J415 than with J591or with 7E11.

In order to assess the specificity of radiolabeled MAb localization inPSMA-positive LNCaP tumors, the uptake of ¹³¹I labeled deJ591 and J415in selected organs was compared to that of an irrelevant IgG antibody.At one day post injection, the tumor uptake (% injected dose/gram) ofboth J415 (12.2±3.24) and deJ591 (8.55+1.29) was significantly highercompared to that of an irrelevant antibody (4.41±0.40). The uptake inlungs, kidney, muscle was similar with all three antibodies. In a secondcontrol study, the tumor uptake of ¹³¹I-deJ591 was determined in nudemice bearing the PSMA-negative prostate tumors (PC3 and DU145). At 4days post injection, the tumor uptake of ¹³¹I-deJ591 was only 0.66±0.07%ID/g in PC3 tumors (n=10) and 0.55±0.03% ID/g in DU145 tumors (n=6). Incontrast, the tumor uptake of ¹³¹I-J591 was 11.4±1.49% ID/g inPSMA-positive LNCaP tumors, significantly greater than in PSMA-negativetumors (p<0.01).

Biodistribution of ¹¹¹In Labeled MAbs

With ¹¹¹In, the tumor uptake of deJ591 and J415 gradually increased withtime and is quite similar to that of 7E11. The kinetics of bloodclearance for both J415 and J591 is faster compared to 7E11. At 6 dayspost-injection, the blood activity of J415 (2.63±0.23) and deJ591(2.52±0.16) is about 50% that of 7E11 (4.16±0.21). As a consequence, thetumor/blood ratios with J415 and deJ591 are significantly highercompared to that of 7E11. There were minor differences in the uptake ofthese three antibodies in liver, spleen and kidney.

The serial gamma camera images of a nude mouse clearly show the intensetumor accumulation of ¹¹¹In-DOTA-deJ591. On day 1, the single tumor (ca250 mg) on the right hind quarter, the blood pool and the liver are wellvisualized. But in the later images, while the activity has cleared fromthe blood pool, the tumor accumulation become gradually more intensecompared to that of liver activity.

Autoradiography

Tumor specimens (n=20) were harvested for hematoxylin (H) and eosin (E)staining and autoradiography to study the intra-tumoral biodistributionof ¹³¹I-labeled MAbs 4 to 6 days after intravenous injection. The H andE stains reveal a considerable amount of necrosis, averaging 50% of thecross-sectional area, in all specimens studied. The autoradiographsreveal a focal, somewhat heterogeneous, distribution pattern with allthree antibodies. Interestingly, the biodistribution pattern with MAbsto PSMA_(int) and PSMA_(ext) reveal almost reciprocal patterns. That is,7E11 (anti-PSMA_(int)) distinctly favors localization to areas ofnecrosis whereas J415 and J591 (anti-PSMA_(ext)) demonstrate a distinctpreferential accumulation in areas of viable tumor. Ex vivoautoradiography, where ¹³¹I-J591 was incubated directly on the tissuesection, demonstrated a homogeneous binding pattern.

Conclusions

The localization of radiolabeled J591 and J415 in PSMA positive LNCaPtumors is highly specific. These results clearly demonstrate thatPSMA-specific internalizing antibodies such as deJ591 and deJ415 may bethe ideal MAbs for the development of novel therapeutic methods totarget the delivery of beta emitting radionuclides (¹³¹I, ⁹⁰Y, ¹⁷⁷Lu)for the treatment of PSMA-positive tumors.

MAbs to PSMA_(ext) had a reciprocal pattern to 7E11, with localizationconcentrated in areas of viable tumor. The inability of 7E11 to targetwell vascularized, viable tumor sites probably explains the inability ofProstaScint® to image bone metastases as well as to explain its failurein RIT trials. By targeting viable tumors, mAbs to PSMA_(ext), likedeJ591 and deJ415, will have a better therapeutic effect. In addition,their ability to target viable tumor imparts better ability to localizewell-vascularized sites in the bone marrow.

Example 3 Animal Studies Using ⁹⁰Y-DQTA-deJ591

In in vitro and in vivo animal models, ⁹⁰Y-DOTA-deJ591 has demonstratedsubstantial anti-tumor activity. In these studies, immunodeficient‘nude’ mice were implanted intramuscularly with PSMA-expressing humanprostate cancer cells (LNCaP). In some studies, the same animals weresimultaneously implanted in the opposite thigh with a PSMA-absent humanprostate cancer line (PC3). Cancers were allowed to ‘establish’ for aperiod of approximately 2 weeks during which time the cancer develops ablood supply allowing further growth. At the time of treatmentinitiation, the cancer implants average 1.0 cm in diameter (orapproximately 5% of the animal's body weight).

Four groups of mice received a single injection of 1.3, 3.7, 5.55 or 7.4MBq of ⁹⁰Y-DOTA-deJ591. At a 1.3 MBq dose level, there was a mixedresponse with minimal reduction in tumor growth rate. However, at dosesbetween 3.7-7.4 MBq, a clear anti-tumor dose-response relationship wasobserved. There was a 30, 55 and 90% reduction in mean tumor volume anda progressive dose-related delay in tumor re-growth of 10, 35 and 60days at 3.7, 5.55, and 7.4 MBq dose levels, respectively. More than 70%of mice that received 3.7-5.55 MBq lived significantly longer than thecontrols and the mean survival time (MST) increased to 80-100 dayscompared to 40 days for controls.

Three groups of mice received 1.11, 2.22 or 3.33 MBq of ⁹⁰Y-DOTA-huJ591every 28 days for 3 doses. At the 1.11 MBq dose level, there was minimaltumor growth during a period of 75-80 days. At 2.22 and 3.33 MBq doselevels, there was a 50-70% reduction in the mean tumor size at day 60followed by gradual increase in tumor size thereafter. The MST increasedby about 200% at 1.11 and 2.22 MBq dose levels compared to control group(120 vs. 40 days).

These studies confirm significant improvement in survival of the⁹⁰Y-DOTA-deJ591 treated animals. The PSMA-absent cancers do not respondto treatment demonstrating the specificity of the treatment.

Example 4 Animal Studies Using ¹⁷⁷Lu-DOTA-deJ591

Nude mice bearing LNCaP tumors treated with ¹⁷⁷Lu-DOTA-deJ591 exhibiteda response similar to the ⁹⁰Y-DOTA-deJ591 treated animals. In this studymice with LNCaP tumors (300-400 mg) were divided into three groups. Acontrol group received no treatment. Group-2 received 200 μCi andGroup-3 received 300 μCi of ¹⁷⁷Lu-DOTA-deJ591. ¹⁷⁷Lu-DOTA-deJ591 was, ina dose dependent manner, able to reduce the mean tumor mass by 80-97% atthe two dose levels. The control group showed a progressive increase intumor size, which was accompanied by a steady loss of body weight andthe mice were sacrificed by fifty-three days because of low body mass.The animals treated with 200 μCi ¹⁷⁷Lu-DOTA-deJ591 had tumor shrinkageup to twenty days post injection, and thereafter tumor regrowth was seenin some animals. The same group also had a nadir in body mass of about90% (of baseline wt) at twenty days post injection, but thereafter asteady rise in the mean body weight to 100-105% of the starting mass. Atninety days, four out of eleven mice had no palpable tumors. The micetreated with 300 μCi of ¹⁷⁷Lu-DOTA-deJ591 had tumor shrinkage up toforty days post injection, and thereafter tumor regrowth was seen insome animals. The same group also had a nadir in body mass of about 90%at twenty days, but thereafter a steady rise in the mean body weight to105-110% of the starting mass. At ninety days, five out of eleven micehad no palpable tumors.

Example 5 Human Trial with ¹³¹I -J591 (Murine), Phase T Clinical TrialTargeting a Monoclonal Antibody (mAb) to the Extracellular Domain ofProstate Specific Membrane Antigen (Pstna_(ext)) in Hormone-IndependentPatients

Hormone-independent patients with rising PSA levels and acceptablehematologic, hepatic and renal function received a single dose of murinemAb J591. Doses are escalated (from 0.5-300 mg) in groups of three tosix patients. Each dose included ≦1.0 mg J591 labeled with 10 mCi ¹³¹Iiodine as a tracer plus “cold” mAb. The dose levels used ranged from 0.5to 300.0 mg as follows: 0.5 mg, 1.0 mg, 2.0 mg, 5.0 mg, 10 mg, 25 mg, 50mg, 100 mg, 125 mg, 150 mg, 200 mg, 250 mg, and 300 mg. Blood and urinewere collected to monitor pharmacokinetics, toxicity and humananti-mouse antibody (HAMA) response. Patients were imaged on the day ofinjection (day 0), as well as days 2, 4 and 6 to track mAb targeting.

Thirty-three patients with hormone-independent prostate cancer wereentered into the phase I biodistribution trial of trace-labeled¹³¹I-J591. These patients received doses ranging from 0.5 to 300.0 mg,in each case conjugated with 1dmCi ¹³¹I. In approximately 80% ofpatients where disease sites had been imaged by conventional studies(CT/MRI and/or bone scan), known sites of prostate cancer metastases canbe imaged. Both soft tissue and bony sites could be visualized on mAbscan. Targeting was specific for prostate cancer sites without apparentlocalization to non-cancer sites. The mean effective serum residencetime of ¹³¹I-mJ591 was determined to be 44.0 hours (median 47.4 hrs).Eight out of sixteen evaluable patients developed a human anti-murineantibody (HAMA) response. That is, their immune systems recognized theforeign nature of the mouse-derived antibody. Only one patient had anadverse event related to the murine antibody. This patient had a severeallergic (anaphylactic) reaction to the murine mAb later determined tobe due to prior (unknown) exposure to murine antibody used inpurification of another experimental drug with which the patient hadpreviously been treated. In sum, thirty-three patients were evaluatedfor targeting: twenty-seven out of thirty-three patients had positivebone scans, with twenty-four out of twenty-seven (about 90%) havingpositive monoclonal antibody scans; nine out of thirty-three patientshad positive soft tissue (CT scans), with eight out of these nine (about90%) having positive monoclonal antibody scans.

Development of a HAMA response, which occurs in most immunocompetentpatients who receive murine antibodies, precludes repeated treatmentswith a foreign species-derived antibody. In order to allow for multipletreatments, murine antibody molecules can be “de-immunized” usingmolecular engineering techniques which remove foreign (mouse) amino acidsequences and replace them genetically with known homologous humansequences. As indicated above, murineJ591 has undergone de-immunizationresulting in “deJ591”.

In conclusion, mAb to PSMA_(ext) targets in vivo specifically todisseminated prostate cancer sites in both bone and soft tissue with nosignificant ‘adverse’ localization and no significant toxicity.

Example 6 Single Human Patient Study Using ⁹⁰Y-DOTA-deJ591

A single patient was treated with mAb deJ591. This patient had bulky,poorly differentiated prostate cancer and had failed multiple courses ofexternal beam radiotherapy as well as multiple forms of hormonal andnon-hormonal chemotherapy. The patient was treated under a singlepatient IND and received a total of twelve doses of deJ591 over a courseof five months, ranging from 10 mg to 200 mg. Four doses (#1, 3, 6 and11) were trace-radiolabeled with either ¹³¹Iodine or ¹¹¹Indium forpharmacokinetic determinations and biodistribution. The mean effectiveserum residence time of ¹³¹I-deJ591 ranged from 31.9 to 51.3 hours,depending on the dose. Tumor localization to known tumor sites wasexcellent after each of the radiolabeled doses over the five monthperiod. Dose twelve was radiolabeled with a therapeutic quantity of⁹⁰Yttrium (19mCi) calculated to deliver less than a 150 rad dose to theblood. This dose was determined in consultation with the FDA and tookinto account prior radiotherapy delivered by external beam and by¹³¹Iodine. No detectable human anti-deimmunized antibody (immune)response developed in this patient. The patient's platelet count droppedto 64,000 (normal: 150,000-300,000) at five weeks post ⁹⁰Y-DOTA-deJ591administration (as an anticipated result of radiation to the bonemarrow) prior to spontaneously returning to normal levels. No otherhematologic or non-hematologic toxicity occurred. The patientexperienced no side effects. The patient's PSA declined from 63 at thetime of deJ591-DOTA-⁹⁰Y administration to 36. No measurable reduction intumor number or size occurred. This patient succumbed to his metastaticprostate cancer ten and one-half months after initiating treatment with⁹⁰Y-DOTA-deJ591.

Example 7 Human Trial with ¹¹¹In-DOTA-deJ591—Phase I Trial of ¹¹¹IndiumLabeled Deimmunized Monoclonal Antibody (mAb) deJ591 to ProstateSpecific Membrane Antigen/Extracellular Domain (PSMAext)

This example describes the results of a clinical trial of deJ591 toassess mAb targeting, toxicity, pharmacokinetics (PK) and immunogenicity(human anti-deimmunized Ab) of this genetically engineered mAb. DeJ591is a strong mediator of antibody dependent cellular cytotoxicity (ADCC).As PSMA is expressed in tumor, but not normal, vascular endothelium ofall cancers, the diagnostic and therapeutic utility of deJ591 may extendbeyond prostate cancer to other cancers.

Patients with recurrent, progressing prostate cancer received fourweekly doses of ¹¹¹In-DOTA-deJ591. Doses were escalated in cohorts ofthree patients and ranged from 62.5-500 mg/m2 (total). Dose level isshown in Table 16. Each dose included 0.02-1.0 mg deJ591 trace-labeledwith 0.1-5 mCi ¹¹¹In via a mAb-DOTA chelate, with the remainder of thedose consisting of unlabeled deJ591. After the first dose, patients wereimaged on the day of injection (day 0) and days 1, 2, 4 and 7.

TABLE 16 ¹¹¹In-DOTA-deJ591 Dosage Loading Dose Maintenance Dose TotalDose Dose Level (mg/m²) (mg/m²) (mg/m²) 1 25 12.5 62.5 2 50 25 125 3 10050 250 4 200 100 500 1-2 mg ¹¹¹In-DOTA-deJ591 (0.2-10 mCi), with thebalance of the dose as “cold” deJ591.

Serum PK, immune reaction and toxicity were evaluated alter each dosefor a minimum of 12 weeks.

Fifteen patients were initially entered in the trial. Thirteen patientsreceived all four planned doses; two patients received ≦1 dose. Onepatient became hypotensive 5 minutes into his first infusion due to arapid infusion rate. The second patient who did not complete treatmentwas withdrawn from the study after one week due to rapid diseaseprogression rendering him no longer eligible. Neither this latterpatient nor the remaining thirteen patients experienced any toxicity orside effects. Ten out of the thirteen patients had positive bone scans;all ten demonstrated excellent mAb targeting to bony sites. Threepatients had soft tissue disease as measured by a CT scan; twodemonstrated mAb targeting to these sites, while the third patient thatwas ¹¹¹In-DOTA-deJ591 negative had a radiated pelvic mass that had notchanged size in >18 months. No mAb targeting to non-prostate cancersites was noted. None of the patients developed an immune reaction tothe antibody. Plasma half-life of deJ591 varied with dose.

In conclusion, deJ591 is non-immunogenic and targets sensitively andspecifically to both bone and soft tissue.

Example 8 Human Trial with ¹¹¹In-DOTA-deJ591, Targeting of HormoneRefractory Prostate Cancer

This example describes the results of a clinical trial of deJ591 toassess mAb targeting, biodistribution, and pharmacokinetics and tooptimize antibody dose for radioimmunotherapy with this mAb in patientsexhibiting hormone refractory prostate cancer.

Twenty-six patients exhibiting hormone refractory prostate cancer wereinjected with a single dose of ¹¹¹In-DOTA-deJ591, consisting of 20 mgdeJ591 labeled with 185 MBq of ¹¹¹In-DOTA. All patients underwent wholebody and SPECT imaging on days 0 (the day of injection), 3, and 6 of¹¹¹In-DOTA-deJ591 injection. In-DOTA-deJ591 imaging results werecompared with CT, MRI, and bone scan. All patients had rising PSA levelson three consecutive measurements.

Twenty-two of the twenty-six patients had imagable disease on routineimaging modalities, while four patients had no imagable disease. Imagingdata revealed ¹¹¹In-DOTA-deJ591 tumor targeting in sixteen of thetwenty-two (72.7%) patients who had imagable disease. Targeting was bestobserved on day 3. No additional (unknown) sites were detected on SPECTimaging. Targeted metastatic sites were in the bone or bone marrow intwelve patients, in the soft tissue in two patients, and in both thebone and soft tissue in two patients.

¹¹¹In-DOTA-deJ591 imaging was false negative in four of the twenty-two(18%) patients who had imagable disease. Non-targeted metastatic siteswere in the soft tissue in three patients and in both the bone and softtissue in one patient.

Accumulation of ¹¹¹In-DOTA-deJ591 in the prostate gland was generallyminimal in patients with intact but irradiated prostate. Amongphysiological sites, highest uptake was observed in the liver, with27+/−1.7% uptake at day 6. The absorbed dose by the liver was 2.8+/−0.25rads/mCi with ¹¹¹In and 20.1+/−2.1 rads/mCi with ⁹⁰Y. Plasma clearance(T1/2) of ¹¹¹In-DOTA-deJ591 was 34+/−5 hours.

¹¹¹In-DOTA-deJ591 specifically targets hormone refractory prostatetumors and is an effective vehicle to target hormone refractory advancedprostate cancer with radioactivity or cytotoxins.

Example 9 Targeting of Hormone Refractory Prostate Cancer with¹¹¹In-DOTA-deJ591 or ¹⁷⁷Lu-DOTA-deJ591

Imaging studies with ¹⁷⁷Lu-DOTA-deJ591, like those described in Example8 for ¹¹¹In-DOTA-deJ591, have produced similar results. The followingsummary represents a combination of the results obtained for ¹⁷⁷Lu and¹¹¹In.

Thirty-nine patients with hormone-refractory prostate cancer have thusfar been enrolled in one of several clinical trials utilizing deJ591.Nuclear imaging with either ¹¹¹In or ¹⁷⁷Lu labeled deJ591 was performedas an initial step of each trial. ¹¹¹In, which is a γ-emitter, was usedeither as tracer for “naked” mAb or as a surrogate imaging agent priorto the administration of ⁹⁰Y (a pure β emitter which does not image onradionuclide scans). ¹⁷⁷Lu emits both γ and β particles, allowing directimaging, and can be used as both an imaging and a therapeutic agent. Theantibody scans were compared to standard imaging studies (bone, CT or MRscans) obtained on each patient to assess the underlying sensitivity,specificity, and accuracy of deJ591 targeting to metastatic sites. Bonyand soft tissue metastases were evaluated independently.

Results have been obtained for bony metastasis in twenty-three patients,and soft-tissue metastasis in twenty-five patients. Fifteen patients hadbone metastases on bone scan, which were accurately targeted by deJ591in twelve patients (80%). In these twelve patients, every cancerouslesion seen on bone scan was identified on the deJ591 scan. All sevenpatients with negative bone scans were negative on the deJ591 scan (100%specificity). For soft tissue metastases, eight out of nine patientswith soft tissue masses on conventional imaging demonstrated accuratetargeting using deJ591 (89%). The one false negative patient hadretroperitoneal adenopathy measuring 8 mm not seen on mAb scan, butwhose bony lesions were all targeted. Fifteen patients withoutdocumented soft tissue metastases had negative mAb scans (100%). Onepatient without soft tissue metastases on initial standard imaging butlesions identified on mAb scan subsequently proved positive on standardimaging. Overall, there was a 91% concordance between standard imagingand mAb scans for soft tissue and bony metastases.

DeJ591 accurately targets known bony or soft tissue metastases in thevast majority of patients. Additionally, one previously unseenmetastatic site was demonstrated on mAb scan and later confirmed with CTimaging. DeJ591 is a highly sensitive and specific agent for targetingmetastatic prostate cancer lesions.

Example 10 Human Trial with ⁹⁰Y-DOTA-deJ591; Phase I Trial ofDe-immunized mAb deJ591-DOTA-⁹⁰Yttrium in Patients with RelapsedProstate Cancer

A phase 1 trial of escalating doses of ⁹⁰Y-DOTA-deJ591 therapy ofpatients with recurrent/relapsing prostate cancer was conducted. Dosesstarted at 5 mCi/m² and were escalated in increments of +2.5-5 mCi/m²for cohorts of three to six patients. The design of this study issummarized as follows: In-DOTA-deJ591 was administered to patients so asto determine the biodistribution of the antibody and the associateddosimetry; and ⁹⁰Y-DOTA-deJ591 was administered 7-10 days later at 5.0,10, 15, 17.5, and 20 mCi/m². All administrations were by intravenousinfusion at a rate of about 5 mg/min. DeJ591-DOTA was labeled at aspecific activity between 3-5mCi-⁹⁰Y/mg antibody to reach the defineddose of ⁹⁰Y, with the balance brought up to 20 mg total deJ591 with“cold” deJ591. Dosages were administered with 6-8 weeks between doselevels. Subsequent doses of ⁹⁰Y-DOTA-deJ591 were allowed.

The subjects for this trial had prostate cancer that had relapsed afterdefinitive therapy (e.g. surgery and/or radiation) and for whom nocurative standard therapy exists.

The objectives of this trial are to: (1) define the toxicity and maximumtolerated dose (MTD) of repeated (fractionated) doses of de-immunizedmonoclonal antibody (mAb) deJ591-DOTA-⁹⁰Yttrium (⁹⁰Y) in patients whohave recurrent and/or metastatic prostate cancer; (2) define thepharmacokinetics of deJ591-DOTA-⁹⁰Y; (3) define the humananti-deimmunized antibody immune response to deJ591-DOTA-⁹⁰Y; and (4)define the preliminary efficacy (response rate) of repeated(fractionated) doses of deJ591-DOTA-⁹⁰Y.

Treatment Protocol:

Patients who developed ≧grade 2 allergic reaction as a result of⁹⁰Y-DOTA-deJ591 would not receive further DOTA-deJ591 and would befollowed for toxicity.

Patients were followed for a minimum of 12 weeks after thedeJ591-DOTA-⁹⁰Y administration. If the patient's disease was stable orresponding at 12 weeks after his last dose, he was followed untilprogression.

The follow-up study consisted of gathering the information shown inTable 17, below, at the indicated times.

TABLE 17 Follow-up Analyses Medical history Day of Rx, week 1, 2, 4, 6,8, and 12, then every 12 weeks thereafter until progression Physicalexam (focused) Day of Rx, week 1, 2, 4, 6, 8, and 12, then every 12weeks thereafter until progression Performance status Day of Rx, week 1,2, 4, 6, 8, and 12, then every 12 weeks thereafter until progressionPSA, PAP, alkaline phosphatase Day of Rx, week 1, 2, 4, 6, 8, and 12,then every 12 weeks thereafter until progression Testosterone Week 4,then every 6 months thereafter until progression Human anti-deimmunizedAb Day of Rx, week 2, 4, 8, and 12 CBC, differential, platelet count Dayof Rx, week 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, and 8, then every 4weeks thereafter until stable, and then every 12 weeks untilprogression. Monitor qod if ANC <1000 and/or platelets <50,000Electrolytes, BUN, creatinine Day of Rx, week 1, 2, 4, and 8, then every4 weeks until stable, and then every 12 weeks until progression Totalprotein, albumin, bilirubin, Day of Rx, week 1, 2, 4, and 8, then every4 weeks until GGTP, AST, ALT, LDH stable, and then every 12 weeks untilprogression Urinalysis Day of Rx, week 1, 2, and 4, then every 4 weeksunless normal or baseline CT or MRI Week 12, then every 12 weeks (ifmeasurable or evaluable disease present at entry and/or if patientclassified as a “responder”) Bone scan Week 12, then every 12 weeks (ifevaluable disease present at entry and/or if patient classified as a“responder”) CXR Week 12, then every 12 weeks (if disease present atentry) Weight Day of Rx, week 1, 2, 4, 8, and 12, then every 12 weeksuntil progression Appetite Day of Rx, week 1, 2, 4, 8, and 12, thenevery 12 weeks until progression Bone pain Day of Rx, week 1, 2, 4, 8,and 12, then every 12 weeks until progression Analgesic intake Day ofRx, week 1, 2, 4, 8, and 12, then every 12 weeks until progression

Pharmacokinetics:

Following injection of ¹¹¹In-DOTA-deJ591, blood samples were obtained at10 min, 1, 2, 4, 24 hours and days 2, 3, 4 and 7. The percent injecteddose (% I.D.) in blood was determined by measuring an aliquot of bloodalong with a known ¹¹¹In standard. Similar blood samples were taken atthe same interval after the ⁹⁰Y-DOTA-deJ591. The % I.D. in blood wasdetermined by measuring an aliquot of blood along with a known ¹¹¹In or⁹⁰Y standard.

Toxicity:

NCI CTEP Common Toxicity Criteria (CTC), version 2 (April, 1999) wasutilized. Since CTEP has standardized the CTC, the NCI does not requireinclusion of the CTC within this document. All treatment areas haveaccess to a copy of the CTC version 2.0. A copy may also be downloadedfrom the CTEP web site.

Definition of Dose Limiting Toxicity (DLT):

Hematologic toxicity: grade 4 granulocytopenia [ANC<500/mm²]; grade 4thrombocytopenia [platelet count<10,000/mm²]; or febrile neutropenia orneutropenic infection as defined by the CTC. Other toxicity: grade≧3non-hematologic toxicity attributable to ⁹⁰Y-DOTA-deJ591.

Definition of Maximum Tolerated Dose (MTD):

The MTD is defined as the dose level at which 0/6 or 1/6 patientsexperience DLT with the next higher dose level having 2 or more patientsexperiencing DLT. Once the MTD has been reached, at least 6 patientsshould be evaluated at that dose to better determine the toxicities ofthe regimen and the pharmacokinetics of ⁹⁰Y-DOTA-deJ591.

Allergic events will be managed as follows: rash, pruritis, urticariaand wheezing will be treated with benadryl and/or steroids as clinicallyappropriate. Anaphylaxis or anaphylactoid signs or symptoms can betreated with steroids and/or epinephrine as clinically indicated.

Specific Interventions Solely for the Purpose of the Study:

Other than the actual administration of deJ591 and related studies todefine the pharmacokinetics and biodistribution of the mAb, the otherinterventions (labs, imaging studies, office visits) performed arestandard procedures. Some of the lab tests would not typically be donein the setting of prostate cancer (e.g., immune reaction levels). Otherlab and radiographic procedures, although standard in the management ofpatients with prostate cancer, may be done at greater frequency thantypical.

Criteria for Therapeutic Response:

Prostate cancer progression is manifest by rising PSA levels, newlesions on bone scan, new disease-related symptoms and, less commonly,increasing size of a measurable soft tissue mass. Response is commonlyassessed either biochemically (PSA change) or by change in size of ameasurable lesion/s.

Biochemical (PSA) response can be determined by comparing the nadir PSAlevel after therapy to the baseline, pre-treatment PSA determined justprior to initiating therapy. A decline of >50% has been demonstrated bynumerous investigators (Petrylak, D. P. et al. (1992) Cancer 70:2870-78;Kelly W. K., et al. (1999) J Clin Oncol 11:607-15; Kantoff P. W., et al.(1999) J Clin Oncol 17:2506-13, 1999; Smith, D. C., et al. (1998) J ClinOncol 16:1835-43) to correlate with improved survival. In addition,Scher, et al (Scher H. I., et al. (1999) MCI 91:244-51) havedemonstrated that a PSA which either declines or shows no increase frombaseline at either 8 or 12 weeks after initiating therapy correlateswith improved survival compared to patients whose PSA rises despitetherapy.

In patients with measurable disease: complete response is defined ascomplete disappearance of all measurable lesions by physical examinationor imaging studies with no appearance of new lesions for ≧2 months.Partial response: is defined as a 50% or greater reduction in the sum ofthe products of the longest perpendicular diameters of all measurablelesions. There may be no new lesions. Stable disease: patients who donot meet the criteria of partial response and who are without signs ofprogressive disease for ≧2 months. Progressive disease is defined as agreater than 25% increase in the sum of the products of the longestperpendicular diameters of the indicator lesions, the appearance of newlesions or a rise in prostate specific antigen above pre-treatmentbaseline.

Duration of response: typically, the first sign of progression will be arise in serum PSA. In this trial the duration of response will be thetime interval from treatment initiation (⁹⁰Y-DOTA-deJ591) untilprogression is documented by either a confirmed rise in PSA, enlargementof the measurable lesion/s, or new lesion/s on imaging studies. Therising PSA must be confirmed by a second, serially rising PSA and theduration will be defined as the time from initiation of treatment to thetime of the first rising PSA.

Results:

Twenty patients have been treated according to this protocol, andnineteen of the twenty can currently be evaluated. All twenty patientshad failed one or more forms of hormone therapy, and eleven of thetwenty patients had failed at least one chemotherapy regimen.Furthermore, the patients all had increasing PSA levels and a minimumplatelet count of 150,000.

Blood chemistry, hematology and PSA levels were monitored for twelveweeks or longer. No significant changes were observed in bloodchemistry, or liver or kidney functions. Hematological changes inplatelets and white blood cell levels were observed at all dose levels.Toxicity was dose-related and limited to reversible myelosuppression(primarily thrombocytopenia). Grade 3-4 thrombocytopenia was observed at15-20 mCi/m². The maximum tolerated dose was estimated to be less thanor equal to 20 mCi/m². ⁹⁰Y-deJ591 radiation dosimetry estimates based on¹¹¹In-deJ591 imaging studies indicate that the organ dose to liver,kidney, spleen, and bone marrow are 20, 19, 18, and 1.7 rads/mCi,respectively. No patients developed an immune reaction.

Dose-related anti-tumor effects were noted. At the first two dose levels(5 and 10 mCi/m²), five out of the eleven (45%) patients had PSA valuesthat continued to increase despite treatment, while six out of theeleven (55%) patients achieved an average 23% reduction in PSA levels.At 15 mCi/m², one out of the four patients (25%) progressed, while threeout of the four (75%) patients achieved an average 25% reduction in PSAlevels. At 20 mCi/m2, all four patients achieved an average 42%reduction in PSA levels. Two of these four patients have PSA declines of70-85% continuing beyond 3 months, as shown in FIGS. 13A and B. Meantime to PSA nadir was 7 weeks post-treatment (range: 2-13 weeks).Measurable responses have also been seen in these patients. The patientin whom the PSA level declined by 85% had 90% shrinkage of multiple softtissue metastases. The patient with the 70% decline in PSA had ameasurable decrease in soft tissue disease of 40%.

Conclusions:

⁹⁰Y-DOTA-deJ591 is non-immunogenic (which would allow for repeatedtreatments) and toxicity has been limited to dose-related, reversiblemyelosuppression. Importantly, ⁹⁰Y-DOTA-deJ591 has dose-relatedanti-tumor effects in patients with advanced prostate cancer. Phase Idata indicates that a single administration of ⁹⁰Y-DOTA-deJ591 (lessthan or equal to 20 mCi/m2) is safe with optimal dosimetry for thetreatment of prostate cancer. In addition, the radiation dosimetryestimates indicate that multiple administrations are also safe.

Example 11 Evidence of PSA Responses in Prostate Cancer PatientsReceiving 90Y-deJ591

Two patients receiving ⁹⁰Y-DOTA-deJ591 had rising PSA levels prior totreatment with radiolabeled J591 (see FIGS. 13A and 13B). The X-axis onthe plots represents time (in days). Negative numbers on this axisindicate days prior to J591 treatment. At the “0” time point, thepatients received ⁹⁰Y-J591 for therapy. The graphs demonstrate that therapidly rising PSA prior to treatment takes a sharp turn within a fewweeks of treatment and becomes stable for a long period of timethereafter (at least ten weeks). The stability of the PSA levelindicates that the progressive disease has stopped progressing. Higherdoses of radiolabeled J591 may lead to a decrease in the disease burdenand/or a prolongation of the cessation of tumor growth rate. Inaddition, repeated doses may also lead to absolute declines in the tumorburden as well as substantial prolongation of cessation of tumor growthrate.

Example 12 Human Trial with ¹⁷⁷Lu-DOTA-deJ591; Phase I Trial ofDe-Immunized mAb deJ591-DOTA-¹⁷⁷Lutetium in Patients with RelapsedProstate Cancer

This example describes a clinical study of subjects who have prostatecancer that has relapsed after definitive therapy (e.g., surgery and/orradiation) and/or who are hormone independent and for whom no standardtherapy exists. There is currently no curative therapy for thesepatients. Furthermore, the example focuses on ¹⁷⁷Lu labeled deJ591. ¹⁷⁷Lu is a both a beta- and a gamma-emitter. As such, it can be used forboth radiotherapy and imaging.

The objectives of this trial were to: (1) define the toxicity andmaximum tolerated dose (MTD) of de-immunized monoclonal antibody (mAb)deJ591-DOTA-¹⁷⁷Lutetium (¹⁷⁷Lu) in patients with prostate cancer whohave recurrent and/or metastatic prostate cancer (Pea); (2) define thepharmacokinetics of deJ591-DOTA-¹⁷⁷Lu; (3) define the biodistributionand dosimetry of deJ591-DOTA-¹⁷⁷Lu; (4) define the humananti-de-immunized antibody (immune) response to deJ591-DOTA-¹⁷⁷Lu; (5)define the preliminary efficacy (response rate) of deJ591-DOTA-¹⁷⁷Lu;and (6) define single and multiple dose schedules for deJ591-DOTA-¹⁷⁷Lu.

Treatment Protocol:

Patients received a dose of deJ591-DOTA-¹⁷⁷Lu administered at aninfusion rate of ≦5 mg/min. The total dose of deJ591 remained fixed at10 mg/m². The ¹⁷⁷Lu dose (in mCi/m²) was escalated in cohorts of threeto six patients for each dose level (see Table 18 below).DeJ591-DOTA-¹⁷⁷Lu was labeled at a specific activity between 3-10 mCi/mgantibody to reach the defined dose of ¹⁷⁷Lu, with the balance brought upto 10/m² mg total deJ591 with “cold” deJ591. Dose escalation waswithheld until at least three patients at the ongoing dose level hadbeen followed for ≧6 weeks without serious hematologic toxicity. If anyof the initial three patients at a dose level experience grade 1 or 2hematologic toxicity by 6 weeks, escalation was withheld until recoverybegan or until 8 weeks of further monitoring and evaluation of toxicityhad occurred. If any patient experienced grade 3 or 4 hematologictoxicity, at least six patients needed to be entered at that dose leveland followed for a minimum of 8 weeks or until recovery begins prior toescalation. If, at any time, two instances of dose-limiting toxicitywere observed at a given dose level, further entry at that dose levelwill be halted. In such a case, at least 6 patients should be entered atthe prior dose level to aid in defining MTD (see “Toxicity” sectionbelow).

Patients who develop ≧grade 2 allergic reaction while receiving ¹⁷⁷Lu-DOTA-deJ591 did not receive further deJ591 and were followed fortoxicity.

TABLE 18 Dose Level Total deJ591* ¹⁷⁷Lu Dose 1 10 mg/m² 10 mCi/m² 2 10mg/m² 15 mCi/m² 3 10 mg/m² 30 mCi/m² 4 10 mg/m² 45 mCi/m² 5 10 mg/m² 60mCi/m² 6 10 mg/m² 70 mCi/m² 7 10 mg/m² 75 mCi/m² 8 10 mg/m² 90 mCi/m² 910 mg/m² 105 mCi/m²  *consisting of deJ591-DOTA-¹⁷⁷Lu at specificactivity between 3-10 mCi/mg with the balance to 10 mg/m² total with“cold” deJ591.

Patients were followed for a minimum of 12 weeks after thedeJ591-DOTA-¹⁷⁷Lu administration. If the patient's disease was stable orresponding at 12 weeks after his treatment, he was followed untilprogression. Patients were considered eligible for retreatment withdeJ591-DOTA-¹⁷⁷Lu at a minimum of six week intervals if hematologicalrecovery was satisfactory.

Except as noted in Table 19, follow-up was as described above in Table174.

TABLE 19 Human anti-deimmunized Ab Day of deJ591-DOTA-¹⁷⁷LuRx, post-Rxweek 1, 2, 4, 8, and 12, then every 12 weeks until progression Chem-7(including Electrolytes, Day of deJ591-DOTA-¹⁷⁷LuRx, post-Rx week 1, 2,4, and BUN, creatinine, glucose) 8, then every 4 weeks until stable, andthen every 12 weeks until progression Liver panel (including: albumin,Day of deJ591-DOTA-¹⁷⁷LuRx, post-Rx week 1, 2, 4, and bilirubin (tot &dir), AST, ALT, 8, then every 4 weeks until stable, and then every 12weeks (alkaline phosphatase) until progression LDH Day ofdeJ591-DOTA-¹⁷⁷LuRx, post-Rx week 1, 2, 3, and 8, then every 4 weeksuntil stable, and then every 12 weeks until progression

¹⁷⁷Lu-deJ591 Imaging:

Total body images were obtained within 1 hour post-infusion (day 0) andat least 5 additional time points in the subsequent 2 weeks (e.g., days1, 3, and 5, 10, 15 or days 2, 4, 6 and 14). The gamma camera imageswere obtained using a dual head ADAC gamma camera fitted with anappropriate collimator. The percent injected dose (% I.D.) in majororgans (heart, liver, spleen, kidneys, bone marrow, GI tract andbladder) was estimated by drawing regions of interest (ROI) anddetermining the relative counts in each organ and kinetics of wash outfrom each organ. SPECT studies were sometimes performed on the abdomen,pelvis and/or areas of suspected metastatic lesions. Where possible,using known standards of ¹⁷⁷Lu, percent injected dose in tumor wasestimated per gram of tumor mass.

Toxicity:

NCI CTEP Common Toxicity Criteria (CTC), version 2 (April, 1999) wasutilized. Since CTEP has standardized the CTC, the NCI does not requireinclusion of the CTC within this document. All treatment areas haveaccess to a copy of the CTC version 2.0. A copy may also be downloadedfrom the CTEP web site.

Three to six patients were entered (or will be entered) at each doselevel. Dose escalation was withheld until at least three patients at theongoing dose level had been followed for 6 weeks without hematologictoxicity. If any of the initial three patients at a dose levelexperience grade 1 or 2 hematologic toxicity by 6 weeks, escalation willbe withheld until 8 weeks to further evaluate toxicity. If any patientexperiences grade 3 or 4 hematologic toxicity, at least six patients hadto be entered at that dose level and followed for a minimum of 8 weeksprior to escalation or until recovery begins prior to escalation. If, atany time, two instances of grade 3 or grade 4 toxicity were observed ata given dose level, further entry at that dose level would beterminated.

Definition of Dose Limiting Toxicity (DLT):

Hematologic toxicity: Grade 4 granulocytopenia (ANC<500/ul) for >7 daysor grade 4 thrombocytopenia (platelets<10,000). Other toxicity: grade≧3non-hematologic toxicity attributable to ¹⁷⁷Lu-DOTA-deJ591.

Adverse Event Definition:

An adverse event is defined as any untoward medical occurrence in aresearch patient during a clinical trial or 4 weeks-posttreatment,regardless of causality. This includes clinical or laboratory findings,inter-current illness or an exacerbation or progression of a disease ora condition present at the time of entry (baseline). An adverse event isnon-serious if it does not meet any of the serious criteria (see below).

Causality/Attribution:

All clinical adverse events and abnormal laboratory values wereevaluated by for potential relationship to the experimental agent. Thefollowing categorizes of causality/attribution will be utilized:definite, probable, possible, unlikely, and unrelated.

Abnormal clinical laboratory values of clinical significance which werepresent at baseline and did not change in severity or frequency duringthe experimental therapy and/or which can reasonably be attributed tothe underlying disease were evaluated by the investigator and recordedin the “unrelated” category. Such events, therefore, were not beconsidered in the evaluation of the safety of this agent.

Preexisting Conditions:

In this trial, a preexisting condition (that is, a disorder presentbefore the adverse event reporting period started) is not reported as anadverse event unless the condition worsens or episodes increase infrequency during the adverse event reporting period.

Adverse Event Definitions:

Each adverse event was classified as serious or non-serious and/expectedor unexpected. An adverse event is classified as serious if it: itresulted in death; it was life-threatening (i.e., the encounteredadverse event placed the subject at immediate risk of death; it does notapply to an adverse event which hypothetically might have caused deathif it had been more severe); it required or prolonged in-patienthospitalization; it resulted in persistent or significant disability orincapacity; and it resulted in a congenital anomaly/birth defect.

Grading:

Toxicity was graded on a scale of 0-4 using either the Common ToxicityCriteria scales or the following:

-   -   (1) 0=no toxicity.    -   (2) 1=mild toxicity, usually transient, requiring no special        treatment and generally not interfering with patient activity.    -   (3) 2=moderate toxicity which may be ameliorated by simple        therapeutic measures; impairs usual activities.    -   (4) 3=severe toxicity requiring therapeutic intervention and        interrupting usual activities. Hospitalization may or may not be        required.    -   (5) 4=life threatening toxicity which requires hospitalization.    -   (6) 5=a fatal toxicity.

Criteria for Therapeutic Response:

Prostate cancer is manifest by rising PSA levels, new lesions on bonescan, new disease-related symptoms and, less commonly, increasing sizeof a measurable soft tissue mass. Response is commonly assessed eitherbiochemically (PSA change) or by change in size of a measurablelesion/s.

Biochemical (PSA) response was determined as described above. Inpatients with measurable disease: Complete response is defined ascomplete disappearance of all measurable lesions by physical examinationor imaging studies with no appearance of new lesions for >2 months.Partial response: is defined as a 50% or greater reduction in the sum ofthe products of the longest perpendicular diameters of all measurablelesions. There may be no new lesions. Stable disease: patients who donot meet the criteria of partial response and who are without signs ofprogressive disease for >2 months. Progressive disease is defined as agreater than 25% increase in the sum of the products of the longestperpendicular diameters of the immeasurable lesions, the appearance ofnew lesions or a rise in prostate specific antigen above pre-treatmentbaseline.

Duration of response: Typically, the first sign of progression will be arise in serum PSA. In this trial the duration of response will be thetime interval from treatment initiation until progression is documentedby either a rise in PSA, enlargement of the measurable lesion/s, or newlesion/s on bone scan. The rising PSA must be confirmed by a second,serially rising PSA and the duration will be defined as the time frominitiation of treatment to the time of the first rising PSA.

Results:

Hormone refractory patients with CT/Bone scan documented prostate cancerlesions and increasing PSA levels were enrolled in a Phase Idose-escalation (10-75 mCi/m²) study. All of the patients had failed oneor more forms of hormone therapy.

To date, twenty-eight patients (seven groups of patients, 3-6/group)have received ¹⁷⁷Lu-DOTA-deJ591 (10 mg/m²). Each group received adifferent dose of ¹⁷⁷Lu-DOTA-deJ591: 10, 15, 30, 45, 60, 70 or 75mCi/m². Blood samples were obtained for two weeks, and imaging studieswere performed five times during the same two weeks. Blood chemistry,hematology, and PSA levels were monitored for three months or longer.Patients having satisfactory hematological recovery after 6 weeks wereeligible for retreatment with deJ591-DOTA-¹⁷⁷Lu.

Imaging studies showed specific tumor localization of ¹⁷⁷Lu-DOTA-deJ591.Four patients had previously unrecognized metastatic foci demonstratedupon 177Lu-DOTA-deJ591 imaging, which was subsequently confirmed byconventional imaging. Imagining of patients treated with¹⁷⁷Lu-DOTA-deJ591 showed targeting of metastatic sites in bone and/orsoft tissue comparable to conventional imaging. Specifically,¹⁷⁷Lu-DOTA-deJ591 had 100% targeting efficacy (15/15) for bonemetastases as compared to conventional imaging and had 80% targetingefficacy (4/5) for soft tissue metastases as compared to conventionalimaging. Thus, overall targeting of metastases for ¹⁷⁷Lu-DOTA-deJ591 was95% (19/20) as compared to conventional imaging. The radiation dosimetryestimates show that the liver is the critical organ receiving7.77+/−2.23 rads/mCi. Dose to bone marrow based on blood activity is1.17+/−0.37 rads/mCi. Plasma T1/2 of ¹⁷⁷Lu-DOTA-deJ591 was 43+/−11hours. No significant changes were observed in blood chemistry, or liveror kidney function. Hematological changes were observed at differentdose levels, but even at the 60 mCi/m² dose level, no serious toxicitywas observed. At 10 mCi/m², none of the patients developedgranulocytopenia or thrombocytopenia. For patients receiving the 15mCi/m² dose level, one patient developed grade 1 thrombocytopenia. Noother significant hematological changes were observed. Of the patientsreceiving the 30 mCi/m² dose level, one patient developed grade 2thrombocytopenia and one patient developed grade 2 granulocytopenia.Otherwise at the 30 mCi/m₂ dose, grade 1 or grade 0 platelet toxicityand neutrophil counts were observed. At 45 mCi/m₂, one patient developedgrade 3 thrombocytopenia, the remaining patients developed grade 2 orgrade 1 thrombocytopenia. In addition, two patients developed grade 2granulocytopenia and three developed grade 1 granulocytopenia or did notdevelop cytopenia at all. For the patients receiving the 60 mCi/m₂ doselevel, one patient developed grade 3 thrombocytopenia and two patientsdeveloped grade 3 granulocytopenia. Otherwise platelet counts andneutrophil counts indicated grade 1 or 2 thrombocytopenia, and grade 1granulocyopenia. Of the six patients treated at the 70 mCi/m₂ doselevel, four patients developed grade 3 platelet toxicity and one patientdeveloped grade 4 platelet toxicity. The remaining patient developedgrade 2 platelet toxicity. In addition, one patient developed grade 4granulocytopenia. The rest of the patients developed grade 2 or grade 3granulocytopenia. At 75 mCi/m₂, all three patients tested developed doselimiting toxicity. Two patients developed grade 3 thrombocytopenia andone patient developed grade 4 thrombocytopenia. In addition, all threepatients developed grade 4 granulocytopenia.

Non-hematologic toxicity observed in the twenty eight patients is setforth in Table 20 below:

TABLE 20 Non-hematologic toxicity Number of Patients with Toxicity GradeAdverse Event 1 2 3 4 Fatigue 12 3 0 0 Anorexia 1 0 0 0 Fever 1 0 0 0Rigors 1 0 0 0 Nausea 5 0 0 0 Vomiting 1 0 0 0 Diarrhea 1 1 0 0Constipation 4 0 0 0 Rash 2 1 0 0 ↑ ALT 7 1 0 0 ↑ AST 11 0 0 0

The first eleven patients that entered the study received a dose of¹⁷⁷Lu-DOTA-deJ591 at levels of 10 mCi/m² (three patients), 15 mCi/m²(three patients), and 30 mCi/m² (five patients). All patients had failedone or more forms of hormone therapy, and one (17%) patient had failedat least one chemotherapy regimen. Toxicity was minimal, with nopatients having grade 2 or 3 adverse events, and limited to reversiblemyclosuppression, primarily thrombocytopenia. No patients developed animmune response to ¹⁷⁷Lu-DOTA-J591.

Among these first eleven patients, dose-related anti-tumor effects werenoted. Following treatment at the 10 mCi/m² dose level, two patients hadPSA levels that continued to rise despite treatment, while the remainingpatient showed stabilization of PSA levels. At 15 mCi/m², two patientshad a mean decrease of 35% in PSA levels, while one patient's PSA levelsprogressed despite therapy. Mean time to PSA nadir was 4 weeks posttreatment (range: 2-6 weeks). One of the three patients that exhibited adecrease in PSA levels, who did not have measurable disease, continuedto have 50% reduced PSA levels even at 18 weeks post-treatment (see FIG.14). One of these patients has been retreated and has received at leastthree doses of ¹⁷⁷Lu-DOTA-deJ591.

Of the total twenty-eight patients, preliminary efficacy results showthat 4 patients experienced at least a 50% decline in PSA levels lasting5 to 8 months prior to returning to pre-treatment values. PSA responseswere seen in an additional 8 patients with PSA declines or prolongedstabilization. In addition, several patients had improvement in pain andKPS. Of the five patients receiving the 30 mCi/m² dose level, four havebeen retreated with ¹⁷⁷Lu-DOTA-deJ591. At the 45 mCi/m² dose level,three out of the five patients have been retreated and at the 60 mCi/m²dose level, two out three patients have been retreated.

Conclusions:

¹⁷⁷Lu-J591 is non-immunogenic (which allows for repeated treatments) andhas low toxicity at doses up to 70 mCi/m². No HAHA response was seen inany of the patients including the retreated patients. ¹⁷⁷Lu-DOTA-deJ591has dose-related anti-tumor effects in patients with advanced prostatecancer. It was found to target prostate cancer metastases in both boneand soft tissue. In contrast to ⁹⁰Y-DOTA-deJ591, which has a maximumtolerated dose estimated to be about 20 mCi/m², ¹⁷⁷Lu-DOTA-deJ591 issafe even at a 30 mCi/m² dose level. At 75 mCi/m², all patientsdeveloped dose-limiting toxicity. Thus, the maximum tolerated dose (MTD)of ¹⁷⁷Lu-DOTA-deJ591 is about 70 mCi/m². Toxicity of ¹⁷⁷Lu-DOTA-deJ591was dose-related and limited to reversible myelosuppression, primarilythrombocytopenia. Non-hematologic toxicity was not dose limiting at anydose level tested. ¹⁷⁷Lu-DOTA-deJ591 appears to eliminate disadvantagesassociated with both ¹³¹I-DOTA-deJ591 (which is dehalogenated in vivoand is not ideal for mAbs that are internalized) and ⁹⁰Y-DOTA-deJ591.The longer residence time of ¹⁷⁷Lu-DOTA-deJ591 in tumor tissue may alsoaugment the anti-tumor response as biochemical (PSA) declines andstabilizations were seen.

Multiple doses, each dose below the MTD of ¹⁷⁷Lu-DOTA-deJ591 have beengiven. With this approach two or more doses have been able to be givensuch that in most cases the cumulative dose delivered exceeds that of asine le dose of ¹⁷⁷Lu-DOTA-deJ591. Multiple dosing may offer severaladvantages. For example, the toxicity from each dose is significantlyless than that experienced after a single dose. The number, andtherefore, the total cumulative dose delivered can be titrated to thedegree of bone marrow tolerance of the individual patient. Unlike asingle dose administration, where in order to avoid excess toxicity tomore than about 17% of the patients-some patients are dosed below theirthreshold, multiple dosing allows patients with more robust marrow toreceive a higher cumulative dose without risking long term compromise.Conversely, those patients with more fragile marrow will not be treatedbeyond their tolerance. It was found that multiple doses of <45 mCi/m²(e.g., 30 mCi/m²) is the best incremental dose. Multiple doses of 30mCi/m² are generally well tolerated. Toxicity at each dose can beobserved and dosing ceased when the toxicity reaches a point (e.g.,grade 3 toxicity) where the next dose would be expected to haveexcessive toxicity. Our data indicates that patients can toleratebetween about 2-5 doses of 30 mCi/m². It is likely that a typicalpatient would get about 3 doses. This would provide a cumulative dose of90 mCi/m², greater than what can be delivered by a single dose withinthe same standards. Some patients with robust marrows may tolerate up toif not more than 5 doses, allowing delivery of more than two-fold thatwhich can be delivered by a single dose. When given multiple doses atincrements of 45 or 60 mCi/m², patients tolerated the 1st dose well, butthe additive effect of the 2nd dose resulted in prolongedthrombocytopenia. Therefore, multiple fractionated doses somewhere inthe range<45 mCi/m² would be acceptable.

Animal data has indicated that a fractionated dose approach provideshigher response rate, longer survival and more cures than a single doseapproach.

Example 13 Imaging Non-Prostate Cancers

In addition to prostate epithelial cells, immunohistochemical studiesshow that PSMA is also expressed by vascular endothelial cells ofnumerous solid tumors, but not by normal vascular endothelium in benigntissues. As discussed above, this expression pattern of PSMA occurs invirtually all solid tumors.

An IRB approved Phase I dose escalation trial of ¹¹¹In-DOTA-deJ591 wasinitiated to assess its value as an therapeutic agent for vasculotoxictherapy, to define its toxicity and maximum tolerated dose, to determineits pharmacokinetics and biodistribution, and to assess forimmunogenicity. Eligible patients were those with refractory solid tumormalignancies whose tumor types are known to express PSMA on theneovasculature.

Fifteen patients received 5 mg (three patients), 10 mg (six patients),or 20 mg (six patients) of ¹¹¹In-DOTA-deJ591, followed by a second dose14 days later.

Patients the participated in the study included eight renal cell cancerpatients, four bladder cancer patients, two colon cancer patients, andone pancreas cancer patient. All patients underwent whole body and SPECTimaging on days 0 (the day of injection), 2, 5, and 7 of¹¹¹In-DOTA-deJ591 injection. The ¹¹¹In-DOTA-deJ591 imaging results werecompared with CT and bone scans.

Imaging data revealed ¹¹¹In-DOTA-deJ591 tumor targeting in ten of thefifteen patients (7 renal cell cancer, 2 bladder cancer, and 1 coloncancer). Targeting was best observed on days 2 and 5, and wasindependent of antibody mass delivered. No additional sites weredetected on SPECT. Targeted metastatic sites included: lungs, femur,retroperitoneal and cervical lymph nodes. Brain metastasis in onepatient (with renal cell cancer) was first detected by ¹¹¹In-DOTA-deJ591imaging and later confirmed by MM. In-DOTA-deJ591 imaging was falsenegative in five of the fifteen patients (2 bladder cancer, 1 coloncancer, 1 pancreas cancer, and 1 renal cell cancer). Non-targetedmetastatic sites included: liver, renal bed, pancreas, lungs, and celiaclymph nodes. Undetected lung lesions measured less than 1 cm in size.

No objective responses occurred, although a colon cancer patient had a50% decline in CEA and two patients had improvement in cancer pain andperformance status.

Two different patients with metastatic kidney cancer, having diseasethat had spread to the lungs, lymph nodes, and/or bones, were injectedwith deJ591 labeled with ¹¹¹Indium. Images of the patients, taken within2 days of injection, demonstrated significant uptake of the antibody inthe known tumor sites in these varying tissues and organs. The extent ofthe uptake of antibody is both substantial and rapid.

Among physiological sites, highest uptake was observed in the liver.Plasma clearance and liver uptake were dependent upon antibody mass;lower mass resulted in faster plasma clearance and higher liver uptake(in terms of percentage uptake). Plasma clearance (T1/2) for 5 mg, 10mg, and 20 mg of deJ591 were 21+/−11 hours, 24+/−6 hours, and 37+/−8hours, respectively. Liver uptake for the same dose levels was28%+/−14%, 17%+/−7%, and 13%+/−5%, respectively.

Based on these data, the protocol was revised to provide dosing for 6consecutive weeks (10, 20, 40 and 80 mg/week dose levels) with theoption for re-treatment on 8 week cycles if patients have stable orresponding disease. To date, nine patients are currently receivingtreatment on this schedule.

¹¹¹In-DOTA-deJ591 specifically targets vascular endothelium of solidtumors. These trials demonstrate that deJ591 is an effective approach totargeting solid tumor vascular endothelium with radioactivity orcytotoxins.

Example 14 Phase II Trial of mAb deJ591 in Combination with Low-DoseSubcutaneous Interleukin-2 in Patients with Recurrent Prostate Cancer

The subjects for this trial have prostate cancer which has relapsedafter definitive therapy (e.g. surgery and/or radiation) and/or who arehormone independent and for whom no standard therapy exists. There iscurrently no curative therapy for these patients.

The objectives of the trial are to: (1) To define the preliminaryefficacy (response rate) of mAb deJ591 in combination with dailylow-dose subcutaneous IL-2 in patients who have recurrent and/ormetastatic prostate cancer; (2) to study the toxicity of mAb deJ591 incombination with daily low-dose subcutaneous IL-2; and (3) to measure invitro the effect of IL-2 on the immune response.

Low-Dose IL-2 Therapy:

IL-2 promotes the proliferation and enhances the secretory capacity ofall major types of lymphocytes, including T cells, B cells, and naturalkiller (NK) cells (Smith K. A. (1988) Science 240:169). The IL-2stimulated expansion of antigen-selected T-cell and B-cell clonesdetermines the magnitude of antigen-specific immune responses, while thequality of the response is determined by IL-2 promoted secretion ofadditional cytokines, cytolytic molecules and antibodies (Smith K. A(1993) Blood 81:1414-1423). In addition, through its effects on NKcells, IL-2 stimulates antigen-nonspecific host reactions that involvean interplay between NK cells and monocytes. As a result of thesefunctions, it follows that IL-2 should be useful as an immune stimulant,particularly in cancer immunotherapy. The therapeutic use of IL-2,however, is made difficult because one of its major effects consists ofthe stimulation of secondary cytokine secretion by IL-2-responsivecells. Many of the potential beneficial effects of IL-2 can beattributed to these secondary cytokines that recruit and activateadditional cell types, especially monocytes, that contribute to thetotal immune/inflammatory reaction. However, these same secondarycytokines, when produced in too large amounts, can also lead to severetoxicity. IL-2 was first used in very high doses for the treatment ofcancer, equivalent to 150 million units (MU) of Chiron Corporation IL-2(Rosenberg S. A (1990) Sci. Am 262:62-69). This high dose was determinedusing the dose-escalation and dose-intensification principles ofchemotherapy, and was associated with significant grade 3 and 4toxicity. IL-2 in high doses is known to cause serious side effectsincluding fever, rigors, malaise, myalgia, nausea/vomiting, hypotensionand possibly death.

In the 1990s, researchers began to examine the immunomodulatory effectsand toxicities of continuous low dose IL-2 (Smith K. A (1993) Blood81:1414-1423; Caligiuri et al. (1991) J Clin Oncol 9:2110-2119;Caligiuri et al (1993) J Clin Invest 91:123-132; Bernstein et al. (1995)Blood 86:3287-3294). These studies demonstrated that doses of IL-2 aslow as 1.2 MU daily resulted in the specific expansion of NK cells withminimal toxicity. Bernstein et al. (1995) Blood 86:3287-3294; Lalezariet al. (2000) HIV Clin Trials). Potential side effects include injectionsite reactions (usually redness at the injection site), asthenia,flu-like symptoms, nausea, diarrhea and eosinophilia. The selectiveexpansion of human CD3⁻, CD56⁺ NK cells during low-dose IL-2 beginswithin 2 weeks of therapy and plateaus after 4-6 weeks of treatment(Smith K. A (1993) Blood 81:1414-1423; Fehniger et al. (2000) J ClinInvest 106:117-124). NK cells can account for as many as 60%-80% ofPBMCs after one month of therapy (Smith K. A (1993) supra). Recentstudies suggest that increased NK cell number results from enhancedNK-cell differentiation from bone marrow progenitors, combined with adelay in IL-2 dependent NK-cell death (Fehniger et al. (2000) supra).The low-dose IL-2 regimens have been specifically designed to completelyavoid toxicity.

Combination Monoclonal Antibody and IL-2 Therapy:

The combination of monoclonal antibodies and IL-2 potentially shouldenhance monoclonal antibody efficacy. IL-2 will function to augment thereticuloendothelial system to recognize antigen-antibody complexes byits effects on NK cells and macrophages. Thus, by stimulating NK cellsto release IFN, GM-CSF, and TNF, these cytokines will increase the cellsurface density of Fc receptors, as well as the phagocytic capacities ofthese cells. Therefore, the effector arm of both the humoral andcellular arms will be artificially enhanced. The net effect will be toimprove the efficiency of monoclonal antibody therapy, so that a maximalresponse may be obtained. A small number of clinical trials havecombined IL-2 with a monoclonal antibody (Albertini et al. (1997) ClinCancer Res 3:1277-1288; Frost et al. (1997) Cancer 80:317-333; Kossmanet al. (1999) Clin Cancer Res 5:2748-2755). In such studies, IL-2 wasadministered intravenously by either bolus or continuous infusion.Toxicity was associated with higher doses of IL-2.

IL-2 Therapy in Prostate Cancer:

A variety of studies have examined the effects of IL-2 on prostatecancer cells in vitro and in prostate cancer animal models, Moody et al.Interleukin-2 transfected prostate cancer cells generate a localantitumor effect in vivo (Prostate, 24: 244-251, 1994; Sokoloff et al.(1996) Cancer, 77: 1862-1872; Triest et al. (1998) Clin Cancer Res, 4:2009-2014; Hautmann et al. (1999) Anticancer Res, 19: 2661-2663; Hillmanet al. (1999) Cancer Detect. Prev. 23: 333-342), although there havebeen few clinical trials of IL-2 in patients with advanced prostatecancer. Hillman et al. (1999) supra; Maffezzini et al. (1996) Prostate,28: 282-286; Morris et al. (2000) Cancer, 89: 1329-1348). A Phase IItrials of I.V. intermediate dose IL-2 (dose ranging from 10-15 MUdaily×4) in patients with hormone refractory prostate cancer wasconducted. In six out often patients, a transient decline in PSA levelswas observed. It is unclear if this was from anti-tumor activity, or ifIL-2 affected PSA expression, as had been reported in vitro studiesusing LNCaP cells (Sokoloff et al. (1996) supra). No regression ofmeasurable disease was observed in any patient. The effect of dailysub-cutaneous low-dose IL-2 in patients with progressive prostate cancerhas not been examined.

Specific Aims:

-   -   (1) To define the preliminary efficacy (response rate) of mAb        deJ591 in combination with daily low-dose subcutaneous IL-2 in        patients who have recurrent and/or metastatic prostate cancer.    -   (2) To study the toxicity of mAb deJ591 in combination with        daily low-dose subcutaneous IL-2.    -   (3) To measure the effect of IL-2 on the peripheral blood        mononuclear cell (PBMC) population.    -   (4) To measure in vitro the effect of IL-2 activated NK cells        from patients on this protocol to induce ADCC with mAb deJ591.

Treatment Protocol:

Patients receive daily low-dose subcutaneous rIL-2 (1.2×10⁶ IU/m²/day)on day 1 through day 56. After three weeks of IL-2 administration,patients receive deJ591 by I.V. (25 mg/m²) once a week for threeconsecutive weeks (on days 22, 29, and 36). IL-2 administration iscontinued during this period another two weeks afterwards. This 8-weekregimen constitutes one cycle of therapy. Patients are evaluated forresponse at the end of one cycle. Patients who have responded to therapyor have stable disease are eligible for additional cycles of therapy.Additional cycles will be initiated when there is at least twoconsecutive rises in PSA at least 2 weeks apart. A three-week lead inwith IL-2 should be sufficient to significantly increase the NK cellpopulation. The dose of deJ591 is based on preliminary pharmacokineticdata from the Phase I ¹¹In-labeled deJ591 trial. The dose of antibodywill be adjusted based on additional data analysis of patients treatedin this manner.

A single cycle of treatment has been initiated. Patients who progress byradiographic documentation after one cycle will be removed from thestudy. Patients who responded by radiographic documentation, or who hadstable disease by imaging with >50% decline in PSA value, or who hadstable disease by imaging with stable PSA levels, will be examined every3-4 weeks. Responding or stable patients will be eligible forre-treatment (a second 8-week cycle) following 2 consecutive PSA risesat least 2 weeks apart, at the discretion of the Principal Investigatorand the option of the patient. Patients who have stable disease byimaging with a rising PSA (>25% of pre-treatment value) will be examinedevery 3-4 weeks. Re-treatment will be at the discretion of the PrincipalInvestigator. In order to be retreated, the patient must have a immunereaction titer< 1/100 and satisfy all initial Eligibility and Exclusioncriteria. Re-treatment will be at the identical dose formulation as theinitial dose given to the patient.

Patient Selection:

The trial is a pilot study to ascertain if low-dose IL-2 in combinationwith deJ591 has activity in prostate cancer. It is possible thatlow-dose IL-2 may have activity in prostate cancer patients. However, ithas been decided not to treat patients with IL-2 alone, or delayantibody therapy, if the PSA declines after 3 weeks of therapy onlow-does IL-2, as it will be interesting to study the combined effect ofdeJ591 with IL-2. If significant activity is observed, a subsequentrandomized trial of IL-2 alone vs. IL-2 with deJ591 will be conducted.It is unclear which stage of patient with prostate cancer might benefitfrom this approach. Therefore, at least ten patients will be treated,each in the following subgroups: 1) Biochemical relapse, hormone naive:rising PSA following radical prostatectomy or radiation therapy, withoutevidence of metastatic disease; 2) Biochemical relapse, hormonerefractory: rising PSA following hormonal therapy without priorchemotherapy (these patients may or may not have radiographic documentedmetastatic disease); and 3) Hormone refractory, having received priorchemotherapy. About 30-40 patients will be enrolled on this trial.

Toxicity:

Toxicity will be scored using the Cancer Therapy Evaluation ProgramCommon Toxicity Criteria, Version 2.0 (April 1999).

IL2 Dosage Modifications:

IL-2 will be permanently discontinued for any study drug-related Grade 4toxicity except hematologic (which can be corrected with EPO or G-CSF orblood transfusions). If at any time during the study, ALT is ≧20 timesthe upper limit of normal, both IL-2 and mAb therapy will be permanentlydiscontinued in that subject. Electrolyte abnormalities that can bereadily corrected will not require permanent drug discontinuation.

This protocol allows for one dose level reduction of IL-2 of 25% to 0.9mU/M². Subjects receiving IL-2 may have their IL-2 interrupted for≧Grade 1 toxicity for 24-48 hours. Toxicities that may cause IL-2 to betemporarily interrupted are:

-   -   Electrolyte abnormalities Grade 1 or higher that cannot be        rapidly corrected;    -   Grade 1 or higher respiratory toxicity;    -   Grade 3 local reaction or any local reaction involving        ulceration;    -   Fever>38° C., or intolerable flu-like symptoms or rigors;    -   Other Grade 3 or greater toxicity, either related or unrelated        to IL-2;    -   Fever suspected to be related to an opportunistic infection;    -   Grade 1 fatigue; and    -   Grade 2 hematologic toxicity that can be corrected with EPO,        G-CSF, or blood transfusions.

Subjects who have their IL-2 interrupted can have it resumed at the samedose or at one dose level reduction within 24-48 hours of stopping drug.The dose of IL-2 for that subject may later be increased to the initialdose at the discretion of the subject and the local investigator.

MAb deJ591:

Allergic events will be managed as follows: rash, pruritis, urticariaand wheezing will be treated with benadryl and/or steroids as clinicallyappropriate. Anaphylaxis or anaphylactoid signs or symptoms will betreated with steroids and/or epinephrine as clinically indicated.Patients will be treated in a general clinical research center equippedfor cardiopulmonary resuscitation.

Both drugs will be discontinued in patients who experience any grade 3or 4 toxicity during the three weeks when mAb is administered. Treatmentwith mAb will resume at a 25% reduction in dose of mAb deJ591 after thetoxicity has returned to grade I or less. If grade 3 or 4 toxicityrecurs on attenuated doses, mAb treatment will be discontinued. IL-2treatment will continue for completion of the 8-week cycle.

Criteria for Therapeutic Response;

Response will be assessed either biochemically (PSA change) and/or bychange in size of a measurable lesion/s using standard response criteriafor prostate cancer (Dawson N. A. (1999) Semin Oncol 26:174-184; Bubley,G. J., et al. (1999) J Clin Oncol, 17:3461-3467)

Biochemical (PSA) response will be determined as described above.Criteria for measuring the disease is as follows: Complete response isdefined as complete disappearance of all measurable lesions by physicalexamination or imaging studies with no appearance of new lesions for ≧2months. Partial response: is defined as a 50% or greater reduction inthe sum of the products of the longest perpendicular diameters of allmeasurable lesions. There may be no new lesions. Stable disease:patients who do not meet the criteria of partial response and who arewithout signs of progressive disease for >2 months. Progressive diseaseis defined as a greater than 25% increase in the sum of the products ofthe longest perpendicular diameters of the indicator lesions, theappearance of new lesions or a rise in prostate specific antigen abovepre-treatment baseline.

Correlative Studies:

Immunologic Phenotyping: Immunofluorescence and phenotyping will beperformed using flow cytometry to measure the PBMC population and toquantify expression of lymphocyte subsets (CD3 for T cells, CD56 for NKcells and CD14 for monocytes) as previously described (Bernstein Z. P.,et al. (1995) Blood 86:3287-3294; Lalezari J. P., et al. (2000) HIV ClinTrials).

⁵¹Cr-Release Assay For Cytolysis By NK/Lak Cells: A standard chromiumrelease assay will be used to measure the ability of NK cells derivedfrom PBMCs to lyse PSMA expressing target cells (Cox J. H., et al.(2000) Mol Biotechnol 15:147-154). NK cells will be isolated from PBMCsusing a series of purifications using Ficoll-HypaquE density gradient,nylon wool columns and negative selection with immunomagnetic beads toremove T lymphocytes and residual monocytes as well as B cells, asdescribed. (Cox J. H. (2000) supra). On the day of the cytotoxicityassay, viable LNCaP cells (target cells) are labeled with no less than100 uCi of ⁵¹Cr for 1 hr in a 37° C., washed, and resuspend in RPMI at aconcentration of 5×10⁴ cells/ml, with a goal of 0.2 to 1.5 cpm per cell.

Additional Assays:

Assays to measure expression of intracellular accumulation of cytokines,such as IL-2, TNF, and IFN, are described in Pala P. et al. (2000) JImmunol Methods 234:107-124. Expression of these cytokines in patientsPBMCs before and on therapy can be measured.

Statistical Methods:

For all outcome variable with measurements taken at various time points,a Repeated Measures Analysis of Variance (RMANOVA) will be carried outto determine any patterns of change over time. Upon finding asignificant time effect, Bonferroni-adjusted painwise contrasts will becalculated to determine which time points differ from one another. Tosimplify the analysis for data obtained at multiple time points, one maychoose to “aggregate” the measurement values taken over time. An areaunder the curve (AUC) analysis for the PSA levels can be carried out.Alternatively, a “slope analysis” may be carried out.

Current Status of Results:

Six patients have been entered and three have completed 8 weeks oftherapy; two patients are being retreated based on PSA stabilization. Athird patient had objective disease progression. Toxicities have beenexpected and minor including fatigue, injection site reactions andasymptomatic thyroid function abnormalities. IL-2 mediated immunemodulation is being evaluated by flow cytometry on peripheral blood toquantify expression of lymphocyte subsets pre- and post-mAb treatment.

Example 15 Conjugation of deJ591 to the Maytansinoid Cytotoxin DM1

This example describes a process for the production of the deJ591-DM1immunoconjugate. The process is based on standard methods known in theart and can therefore be generalized to other antibodies, includingother antibodies of the invention such as deJ415.

The methods of conjugation are based on several small scale experiments,including one experiment performed using 5 g of deJ591 starting material(Lot 1552-60S) and three experiments performed using between 6.7 g and7.3 g of deJ591 starting material (Lots 1552-168, 1552-104, and1610-036).

The steps involved in the methods of conjugation are as follows:

-   -   1) 5 g to 7.5 g of deJ591 antibody is concentrated by tangential        flow filtration (10 kD NMWCO membranes) to 25-30 mg/ml and        diafiltered against 5 volumes of 50 mM potassium phosphate, 2 mM        EDTA, pH 6.0. The yield is typically between 98% and 100%.    -   2) The concentrated antibody is filtered through a 0.2μ filter,        if opalescent, and then modified with N-succinimidyl        4-(2-pyridyldithio) propionate (SPP) at a concentration of 20-22        mg/ml antibody and about 7 molecules of SPP per molecule of        antibody; preferably, 6.3 molecules of SPP per molecule of        antibody are used, 6, 5, 4, or any fraction thereof can also be        used. The modification is done in 50 mM sodium citrate or,        preferably, potassium phosphate, 2 mM EDTA, 5% ethanol, pH 6.0,        for 2.5+/−0.5 hours. The modification vessel is a 500 ml round        bottom flask.    -   3) The SPP-modified antibody is separated from the reaction        mixture of step 2) using gel filtration chromatography and a        Sephadex G-25™ column. The column load represents about 25% of        the column volume and the chromatography is done in 50 mM        potassium phosphate, 2 mM EDTA, pH 6.0, at a flow rate of 50        cm/hr. The modified antibody elutes between 38-75% column        volume. Typically the yield of this step between 95% and 100%        and the SPP to antibody ratio is about 5.4 to 5.9 SPP        molecule/antibody.

4) At a concentration of about 10 mg/ml, the SPP-modified antibody isconjugated with DM1 (using 1.7 molecules of DM1/molecule of SPPconjugated to the antibodies) for 20+/−4 hours. Typically, the reactiontime is between 16.25 and 17.7 hours and is carried out in a 1 L roundbottom glass flask equipped with a magnetic stirring bar. Theconjugation reaction is done in 10% EtOH or more preferably in 3% DMA,with 10% sucrose (100 mg sucrose/ml of reaction). At the end of thereaction the conjugated antibody is filtered through a 2.0μ filter and aspectrophotometry reading is taken.

-   -   5) The conjugated antibody is separated from unreacted DM1 by        gel filtration chromatography using a Sephadex G-25™ column. The        column load represents 22-23% of the column volume and the flow        rate is about 50 cm/hr. The column is equilibrated and run in 20        mM succinate, 5% or 10% sucrose, preferably 10% sucrose (100        mg/ml), pH 5.5. The antibody conjugate elutes between about 31%        and 65% of column volume, and is collected from the start of the        peak elution to the start of the peak trailing edge as a single        fraction, followed by fractionation of the remaining peak        material in 15×2% column volume fractions. All fractions are        adjusted to 100 mg/ml of sucrose (10% sucrose) through the        addition of appropriate amounts of 50%) sucrose. The 2% column        volume fractions are assayed by analytical sizing (TSK 3000SWL)        and selected fractions (fractions 1 and 2) are pooled together        with the main peak. The fractions are assayed using analytical        sizing with the pooling criterion being the 24 minute peak        representing <20% or the total peak area. Typically the yield of        this step is between 60% and 65% with the exception of run        1552-104 where there was no sucrose present in the reaction        and/or purification mixture. The eluted antibody concentration        ranges from 3.8 to 4.2 mg/ml and the ratio of DM¹/antibody        ranges from 3.6 to 3.9.    -   6) The antibody conjugate is then concentrated to 7-10 mg/ml        using a 10 kD NMWCO tangential flow filtration membrane and        diafiltered against 5 volumes of 50 mM succinate, 10% sucrose,        pH 5.5 (Inlet Pressure<10 psi). Following diafiltration the        antibody conjugate is adjusted to 5 mg/ml. Typical yield for        this step is between 92% and 100%, with the final protein        concentration being between 4.85 and 5.1 mg/ml.    -   7) Finally, the antibody conjugate is filtered through a 0.2 m        filter and aliquoted to the specified volumes. Step yield is        between 90% and 100% and the final DM1-antibody ratio is 3.5 to        3.8.

The resulting deJ591-DM1 conjugates were analyzed according toappearance, concentration, DM¹/antibody ratio, endotoxin, non-specificcytotoxicity, acetone extractable DM1, analytical sizing, reduced andnon-reduced SDS-PAGE, pH, bioburden, specific cytotoxicity, and IEF.Selected analytical results for lots 1552-168, 1552-104, and 1552-036are shown in Table 21, below.

TABLE 21 Amount Final Recovered Concentration DMl/ Process Lot No.(mg/ml) (mg/ml) antibody Recovery (%) 1552-168 3.85 5.1 3.7 57.21552-104 2.75 4.85 3.5 37.8* 1610-036 3.41 5.05 3.8 47.2 Mean 3.34 5.003.67 47.4 Standard Dev. 0.55 0.13 0.15 9.7 % c.v. 16.6 2.6 4.2 20.5*Lower recovery due partly to the lack of sucrose in the conjugationreaction and the second G-25 gel filtration run and partly to the factthat the front end of the product peak was not collected due to amalfunction in the chart recorder.

Briefly, the process can be summarized as follows.

-   -   1. Concentration of deJ591 using Tangential Flow Filtration        (TFF).    -   2. The antibody is then modified with TPA by addition of SPP        reagent.    -   3. Single modified antibody fractions are then separated from        the reaction mixture on a Sephadex G25F SEC chromatography        column.    -   4. The antibody is conjugated with DM1.    -   5. The conjugated antibody is then purified on a Sephadex G25F        SEC chromatography column, and pooled.    -   6. The conjugated antibody is then concentrated and buffer        exchanged into formulation buffer by TFF.    -   7. Finally, the final concentration is adjusted to about 5.0        mg/mL. The overall process yield, as assessed by recovery of        antibody is typically about 60%, but can vary greatly depending        on the molar ratio of SPP linker to antibody used. For example,        typical percent yields using a 7:1 linker to antibody ratio are        about 76% (with a DM1:antibody ratio of about 3.5:1); for a 6:1        ratio, about 76% yield (with DM1:Ab of 3.6:1); 5:1 yields about        88% (with DM1:Ab of 3.2:1); and 4:1 yields about 92% (with        DM1:Ab of 2.6:1).

Example 16 Method of Manufacture of deJ591 Harvest/Clarification andConcentration

A 2000 L scale production fermentation reaction mixture was harvested bycentrifugation. Following centrifugation the harvest supernatant waspassed through a depth filter to remove any remaining cell debris. (The200 L scale process utilized depth filtration alone to remove cells andrelated debris.) The resultant clarified harvest was concentrated usingan ultrafiltration system, such as a 0.2 μm system.

Filtration

The concentrated product was aseptically filtered and stored at 2 to 8°C.

Purification

A brief summary of the purification process is given below. At the endof each purification processing step and at the time of dispensing intothe bulk purified product container, the in-process product was 0.2 μmfiltered. In-process product was stored at 2 to 8° C. between processingsteps.

Purification By Recombinant rmp-Protein A Chromatography

The immobilized recombinant rmp-Protein A chromatography step was run asa multi-cycle process. The recombinant rmp-Protein A used forpurification was dedicated to deJ591.

The processed harvest supernatant was divided into appropriately sizedaliquots and loaded onto the recombinant rmp-Protein A column insuccessive cycles. Each aliquot was processed as follows: The Protein Acolumn was equilibrated using 50 mM Glycine Glycinate pH 8.0 buffercontaining 250 mM Sodium Chloride. Processed harvest supernatant wasadjusted to pH 8.00±0.5 using 1.0 M Tris base prior to being loaded ontothe column. After loading the column, the unbound impurities were washedthrough the column using 50 mM Glycine Glycinate pH 8.0 buffercontaining 250 mM Sodium Chloride, followed by 9 mM sodium formate pH6.0 buffer. Bound antibody was eluted from the column as a singlefraction using 13 mM Sodium Formate, pH 4.0 buffer. Bound impuritieswere eluted from the column using 100 mM Citric Acid, pH 2.1 betweencycles. Upon completion of the chromatography step or after 5 cycles thecolumn was cleaned with a solution of 6 M Guanidine HCl.

The recombinant rmp-protein A matrix was removed from the column andstored in 20% ethanol between batches.

pH 3.7 Treatment

The recombinant rmp-Protein A column eluate from each cycle wascollected and the pH immediately adjusted to 3.70±0.10 with 2.0 M aceticacid. The eluate was held at this pH for a minimum of 30 minutes and amaximum of 45 minutes. The pH of the eluate was then readjusted to pH7.50±0.20 using 2.0 M Tris hydrochloride pH 9.0 buffer.

Concentration and Diafiltration

The in-process product was concentrated using a dedicatedultrafiltration unit.

Purification by Q Sepharose Anion Ion-Exchange Chromatography

The Q Sepharose anion ion-exchange chromatography step was a multicycleprocess.

Virus Reduction Filtration

The in-process product was filtered through a Pall Ultipleat AB virusreduction cartridge filter.

Concentration and Diafiltration

In preparation for SP Sepharose chromatography, the in-process productwas concentrated and subsequently diafiltered into 25 mM Sodium AcetatepH 5.0 buffer containing 25 mM Sodium Chloride.

Purification by SP Sepharose Cation Ion-Exchange Chromatography

The SP Sepharose cation ion-exchange chromatography step was amulticycle process. The SP Sepharose resin was used for the productionof one product batch and was then discarded.

The SP Sepharose cation exchange load was divided into appropriatelysized aliquots for each cycle. Each aliquot was processed as follows:The chromatography column was equilibrated using 25 mM Sodium Acetate pH5.0 buffer containing 25 mM Sodium Chloride. An aliquot was loadeddirectly onto the column. Unbound impurities were washed through thecolumn using equilibration buffer. Bound antibody was eluted from thecolumn using 25 mM Sodium Acetate pH 5.0 buffer containing 115 mM SodiumChloride. The column was regenerated between cycles using 25 mM SodiumAcetate pH 5.0 buffer containing 2 M Sodium Chloride. The eluate fromeach cycle was analyzed by SDS PAGE. Cycles showing similar purities byvisual comparison of SDS PAGE gels were pooled.

Final Concentration Adjustment and Diafiltration

The in-process product was concentrated and diafiltered into finalformulation buffer (50 mM Sodium Phosphate pH 5.5 buffer containing 100mM Sodium Chloride and 2 mM EDTA).

0.2 μm Final Filtration and Dispensing

The purified product was 0.2 μm filtered and aseptically dispensed intoautoclaved polypropylene containers.

Example 17 Determining the Ratio of DM1:deJ591

The DM1/deJ591 ratio was determined by measuring the total DM1 molarconcentration spectrophotometrically and dividing by the molardeJ591-DM1 concentration [C_(CJ)] calculated as described above. Themolar concentration of DM1 was based on the absorbance at 252 μm and wascorrected for the contribution of deJ591 antibody to the OD²⁵² using avalue of 0.378 for the ratio of the deJ591 antibody molar extinctioncoefficient at 252 nm to that at 280 nm. The DM1 concentration wascalculated from the absorbance at 252 nm and 280 nm according to theequation below using the corrected OD²⁵² and the molar extinctioncoefficient for DM1 [ε₂₅₂=26,790 M⁻¹].

$C_{{DM}\; 1} = \frac{\left( {A_{252} - \left( {A_{280}*\left( \frac{ɛ_{{ab}@252}}{\underset{\_}{ɛ_{{ab}@280}}} \right)} \right)} \right)}{ɛ_{{DM}\; {1@252}} - \left( {ɛ_{{DM}\; {1@280}}*\left( \frac{ɛ_{{ab}@252}}{\underset{\_}{ɛ_{{ab}@280}}} \right)} \right)}$$\frac{{DM}\; 1}{ab}\mspace{14mu} {ratio}\mspace{14mu} \frac{{CDM}\; 1}{2!}$C_(DM 1) = Molar  concentration  of  DM 1ɛ_(ab):  molar  extinction  coefficient  of  antibodyɛ_(DM 1)  molar  extinction  coefficient  of  DM 1A  absorbanceC_(cj) = Molar  concentration  of  deI 591-DM 1  proteinNumbers  denote  wavelength

The specification for DM1/deJ591 ratio was 3.0-4.0. The average valueover the 4 non-GMP and 2 GMP batches was 3.55. Our manufacturing historyand process development experiments have indicated that 3.5 is anoptimal target ratio. The specification has been set based on a 3.5standard deviation window around this target ratio.

Example 18 Use of the mAbs for Targeted Delivery of Cytotoxic Drugs toProstate Cancer Cells

Anti-PSMA antibodies can be conjugated to substances with high cytotoxicpotential, such as drugs of the maytansinoid class. Maytansinoids exerttheir cytotoxic effects by interfering with the formation andstabilization of microtubules. They have 100- to 1000-fold greatercytotoxic potential than conventional chemotherapeutic agents (such asdoxorubicin, methotrexate, and Vinca alkaloids) (Chari, R. V. J. et al.(1992) Cancer Res. 52: 127).

Both murine and deimmunized J591 antibodies have been conjugated to themaytansinoid, DM1, via a hindered disulfide bond. This bond is cleavedintracellularly allowing release of the drug. One or more lysineresidues in the constant regions of the antibodies were conjugated to alinker containing a pyridyldithio group, which was, in turn, coupled toa maytansinoid toxin. A ratio of 3 to 4 moles of maytansinoid per moleof IgG is preferred.

The process for the DM1-linked J591 antibodies starts by reacting J591with a linker that contains both a pyridyldithio group and aN-hydroxysuccinimide leaving group. In this case, the linker wasN-succinimidyl 4-(2-pyridyldithio) propionate (or SPP), although otherlinkers can be used. The products of the reaction include modified J591antibodies that contain one or more linker groups(4-(2-pyridyldithio)propionone) attached to surface exposed lysinegroups, with the linker groups retaining the pyridyldithio reactivegroups, and N-hydroxysuccinimide leaving groups. The J591 antibodies arethen separated from the reaction mixture and N-hydroxysuccinimide by gelfiltration, e.g., using sephadex G25. The modified J591 antibodies arereacted with DM1, which contains a thiol group that reacts with thepyridyldithio groups now present on the surface of the modifiedantibody, thereby producing J591-DM1 immunoconjugates and thiopyridine.The J591-DM1 immunoconjugate is isolated from the reaction mixture andthiopyridine by size exclusion chromatography, e.g., using a sephacrylS300 column. Methods for preparing maytansinoid conjugates are describedin U.S. Pat. Nos. 5,208,020; 5,475,092; 5,585,499; 5,846,545; and6,333,410, the contents of which are incorporated by reference.

A study evaluating the efficacy, toxicity, and antigen selectivity ofthe murine J591-DM1 immunoconjugate in the treatment of prostate cancercells in vivo is described below.

Experiment 1 Establish Efficacy, Toxicity, and Dose-Response CurvesIn-Vivo

Tumor xenografts of LNCaP cells were established in the right flank ofBALB/c mice (5×10⁶ cells injected subcutaneously). Animals were observeduntil tumors were visible (7-10 mm). Animals were then treated with PBS,unconjugated mAb J591, unconjugated DM1, both mAb J591 and DM1 butunconjugated, and J591-DM1 immunoconjugate (100, 200, 300, and 400mcg/day intraperitoneal qday×5 days). The mAb J591 was modified tointroduce dithiopyridyl groups and then conjugated to DM1 via a hindereddisulfide bond, as described above. Unconjugated mAb J591 and DM1 weregiven in equimolar concentrations.

Experiment 2 Selectivity for PSMA-Positive Tumors

Tumor xenografts of both LNCaP cells (5×10⁶ cells injectedsubcutaneously in the right flank) and PC3 cells (3×10⁶ cells injectedsubcutaneously in the left flank) were established in BALB/c mice.Animals were observed until LNCaP tumors were visible (PC3 tumors werepresent but much smaller). Animals treated with J591-DM1immunoconjugate: 300 mcg/day intraperitoneal qday×5 days

Experiment 3 Determine Optimal Dosing Schedule for the Immunoconjugate

Tumor xenografts of LNCaP cells were established subcutaneously in theright flank of BALB/c mice. When tumors were visible, animals weretreated with PBS, unconjugated mAb J591, J591-DM1 immunoconjugate (300mcg/day intravenously qday×5 days; one course given), J591-DM1immunoconjugate (400 mcg/day intravenously every other day for 5 dosesin 10 days; one course given), and J591-DM1 immunoconjugate (300 mcg/dayintravenously qday×5 days; two courses given for a total of 10 doses).For animals receiving 2 courses of J591-DM1 immunoconjugate, the second5-day course was given at tumor volume nadir (typically seen on days21-24 after completion of the first course).

All animals were photographed pre- and post-treatment and tagged foridentification. Tumor volume was determined (length×width×depth) andclosely followed. Animal weight was followed as a measure of toxicity.

Results Experiment 1

All mice treated with unconjugated DM1 (either alone or withunconjugated mAb J591) died within 48 hours of treatment completion.Immunoconjugate at doses of 400 mcg/day was lethal to 50% of the mice.With doses of 300 meg/day, significant reductions in tumor volume werenoted with several complete responses and minimal toxicity. Thesecomplete responses were not durable, however, with subsequent increasesin tumor size noted 2-20 days after tumor volume nadir.

Results Experiment 2

In mice with both LNCaP and PC3 tumors, LNCaP tumor volume decreasedsubstantially after treatment with the J591-DM1 immunoconjugate.Although complete responses were not achieved in these animals, tumorvolume nadirs averaged 0.08 cm³. PC3 tumor growth was not affected bythe J591-DM1 immunoconjugate as noted by a steady increase in PC3 tumorvolume after treatment.

Experiment 3

Although route of administration was different (intravenous vs.intraperitoneal), treatment with one course of J591-DM1 immunoconjugateparalleled the results obtained from Experiment 1. Complete responseswere achieved (at days 21-24) but these results were not durable. Inanimals treated with every other day dosing of 400 mcg, toxicity wasdemonstrated (weight loss>10% of total body weight) but all animalseventually survived. Tumor volume decreased in all animals but completeresponses were not achieved. All animals treated with 2 courses ofJ591-DM1 immunoconjugate have achieved complete responses lasting atleast through day 66.

Conclusions:

Murine J591 anti-PSMA antibody can be conjugated effectively to drugs ofthe maytansinoid class (such as DM1). These J591-DM1 immunoconjugatesprovide highly selective, antigen-specific targeted delivery of thiscytotoxic drug to PSMA-positive prostate cancer cells in-vivo. Thegreatest reduction in tumor volume with minimal toxicity was noted at adose of 300 mcg/day. The optimal dosing schedule appears to be two 5-daycourses of J591-DM1 immunoconjugate with the second course given attumor volume nadir (day 21-24).

Example 19 Pharmacodynamics and Efficacy of the deJ591-DM1Immunoconjugate

In parallel to the experiments described in Example 16, additionalexperiments were performed with the deJ591-DM1 immunoconjugate. Theexperiments and results obtained therefrom are described below.

In Vitro Pharmacodynamics of deJ591-DM1 and DM1:

Experiment: LNCaP cells were plated into 96 well plates at an initialdensity of 1000 cells per well for a proliferation assay. deJ591-DM1 orDM1 was added to the wells over a range of concentrations (0.01 nM to100 nM) and left in contact with the cells for defined periods of time(0.5, 2, 8, 24, 72 and 144 hours). At the end of that period the drugcontaining media was removed and replaced with drug free media until atotal of 144 hours elapsed from the start of the experiment. Theresulting effect of drug exposure on proliferation was then determined.

Results: The pharmacodynamic analysis using the methodology developed byKalns et al. (1995), Cancer Research 55:5315-5322, the contents of whichare incorporated herein by reference, indicates that when consideringthe relative importance to observed effect from the variables (1) timeof exposure and (2) concentration, that exposure time is more importantfor deJ591-DM1 compared to DM1.

Serum Levels of DEJ591-DM1 in Mice after a Single IV Dose

Experiment: Mice were administered an intravenous 14.5 mg/kg dose ofdeJ591-DM1 and sera samples collected for analysis of the test articleusing an ELISA assay for human antibody. Samples were collected fromgroups of 3 mice at 6, 24, 48, 72 and 168 hours. Data were fit to abi-exponential equation to determine pharmacokinetic parameters. Theseparameters were used to simulate serum levels after multiple dosing.

Results: Analysis of the pharmacokinetic data indicated a serum halflife of deJ591-DM1 in mice as measured in the ELISA assay, to be 130hours.

deJ591-DM1 Serum Levels in C.B-17 Scid Mice With or Without thePSMA-positive CWR22 Xenograft Tumor

Experiment: Non-tumor-bearing mice were dosed intravenously (IV) via thetail vein with a dosage of 14.56 mg/kg deJ591-DM1 (Lot No. 1552-39,equal to 240 μg/kg DM1-equivalents) and the tumor-bearing mice weredosed with 12.93 mg/kg deJ591-DM1 (Lot No. 1552-60S, equal to 240 μg/kgDM1-equivalents). Blood samples were collected at 6, 24, 48, 72, and 168hours post dosing for the non-tumor-bearing mice and at 6, 24, 72, 168,336, and 504 hours post dosing for the CWR22 xenograft-bearing mice.Serum was analyzed quantitatively for the presence of humanimmunoglobulin G (IgG) using an enzyme linked immunosorbent assay(ELISA) for human IgG. This assay measures only human IgG and providesno information regarding the amount of DM1 present either conjugated tothe antibody or released unconjugated into the serum. The data for serumconcentrations of IgG were fit to a standard bi-exponential equationusing nonlinear regression to provide an estimate of the terminalhalf-life for the circulating human IgG.

Results: In the non-tumor-bearing mice, higher serum concentrations forhuman IgG were observed and the terminal half-life in serum wasestimated to be approximately 5 days (120 hours). In these mice, themaximal concentration of 170 μg/mL IgG (2,800 ng/mL DM1-equivalents ifall IgG was deJ591-DM1) was measured at the first sample point of 6hours post dosing, decreasing to 77 μg/mL from the sample at 24 hourspost dosing. In the CWR22 tumor-bearing mice, the maximum IgGconcentration was 74 μg/mL (1,373 ng/mL DM1-equivalents if all IgG wasdeJ591-DM1), at the first sample point of 6 hours, decreasing to 36μg/mL from the sample at 24 hours postdosing. The estimated terminalhalf-life (240 μg/mL DM1-equivalents) for the IgG in serum oftumor-bearing mice was approximately 8.4 days (202 hours). Thus, singleadministration of deJ591-DM1 in tumor-bearing mice produced serumconcentrations (1,373 ng/mL DM1-equivalents) likely in excess of thoserequired to inhibit PSMA-positive cells in tissue culture (for example,IC50 on PSMA-positive LNCaP cells=210 ng/mL deJ591-DM1 with 6-dayexposure in tissue culture.

In a subsequent pharmacokinetic study of deJ591-DM1 in immunocompetent,nontumor-bearing, male CD-1 mice, the maximal concentration of theanalyte deJ591-DM1 after a single IV administration of 10 mg/kg was 196μg/mL (3,528 ng/mL DM1-equivalents, assuming 3.7 DM1/J591). A terminalelimination half-life (t_(1/2)) of approximately 21 hours was determinedfor the analyte deJ591-DM1, with a considerably (2-fold) longer t1/2 of46 hours for total deJ591 (representing intact deJ591-DM1 anddeconjugated deJ591). As one theory, not meant to be limiting, thedifferences in apparent half-lives (120 hours for human IgG innontumor-bearing SCID mice versus 46 hours for total deJ591 in CD-1mice), may be related to differences in the strain (immunocompetency) ofthe mice and/or to differences in the assays used to quantitate antibodylevels.

Effect of Dose Interval on Efficacy Against CWR22 Prostate Xenografts

Experiment: Male SCID mice were implanted by serial passage of CWR22prostate tumor xenograft. When these tumors reached 200-250 mm³ size(estimated from external caliper measurement), mice were randomized intotreatment groups of 8 to receive vehicle only (every 7 days) ordeJ591-DM1 at a dose of 14.5 mg/kg antibody conjugate (equivalent to 240ug/kg DM1) given on one of the following schedules (every 7, 14, 21, or28 days). All treatments were given intravenously. Tumor growth andanimal health were continually monitored throughout the study with tumorgrowth measured every 3 days.

Results: Differences in the schedule of administration have distincteffects on the observed tumor growth for the CWR22 xenograft in SCIDmice. The tumor growth can be characterized as slowed growth for theschedule of 21 or 28 day dosing interval until they reached the maximumsize permitted under our IACUC regulations. For the 7 and 14 day dosinginterval schedule, there is an apparent block in tumor growth with aresumption of normal growth kinetics approximately 30 days after thelast dose. The results when considered with the pharmacokineticsimulation suggest a relationship between duration of exposure andmaintaining a minimum effective concentration.

deJ591-DM1 Efficacy in PSMA-Positive CWR22 Xenografts: Dosage andSchedule: Comparison I

Experiment: Male C.B-17 SCID mice bearing CWR22 xenografts approximately200 mm³ in size received IV injections (200 μL constant volume) of thetest articles according to the dose and schedule as shown in Table 22.

TABLE 22 Dosage, Schedule and Response of Scid Mice Bearing CWR22Xenografts: Comparison I DM1- equivalents Tumor Growth Test ArticleDosage (μg/kg) Schedule Delay^(a) (Days) Vehicle 0 0 qdX5 0 Maytansine240 μg/kg 240 qdX5 17.7 dcJ591-DM1 7.28 mg/kg 120 qdX5 19.6 deJ591-DM114.56 mg/kg 240 qdX5 26.9 deJ591-DM1 7.28 mg/kg 120 q3dX5 27.1deJ591-DM1 14.56 mg/kg 240 q3dX5 36.7 ^(a)Tumor growth delay is thedifference in time (days) for the treatment group to reach 1000 mm³compared with the vehicle-treated group, calculated from the meanvalues.

Results: deJ591-DM1 exhibited a substantial tumor growth inhibitoryeffect (see tabulated tumor growth delay), but was non-curative for theCWR22 xenograft at the doses and schedules tested. Tumor growthinhibition curves for maytansine and deJ591-DM1 are shown in FIGS. 16Aand 16B.

Immunohistochemical analysis of tumors posttreatment showed equivalentPSMA expression compared to vehicle-treated controls. In conclusion,deJ591-DM1 produced a dosage- and schedule-dependent inhibition of CWR22prostate cancer xenograft growth that was greater than equimolar dosagesof maytansine alone. The associated toxicity was less for all dosages ofdeJ591-DM1relative to maytansine alone.

deJ591-DM1 Efficacy in PSMA-positive CWR22 Xenografts and Effect onSerum PSA Levels: Dosage and Schedule: Comparison II

Experiment: The PSA-secreting, PSMA-positive CWR22 xenograft was used tostudy the following: (i) the relative effect of the deJ591-DM1constituent elements (deJ591 and DM1) on the CWR22 xenograft growth,(ii) the influence of dosing interval on efficacy of deJ591-DM1, (iii)the dosage-response relationship of deJ591-DM1 on a q3dX5 schedule, (iv)the CWR22 tumor response to a second course of deJ591-DM1 treatment, and(v) the relationship between tumor response to deJ591-DM1 and serum PSAlevels. Male C.B-17 scid mice bearing CWR22 xenografts approximately 200mm³ in size received IV injections (200 μL constant volume) of the testarticles according to the dose and schedule shown in the Table 23.

TABLE 23 Dosage, Schedule and Response of Scid Mice Bearing CWR22Xenografts: Comparison II DM1- equivalents Tumor Growth Test ArticleDosage (μg/kg) Schedule Delay^(a) (Days) Vehicle 0 0 q3dX5 0 DM1 240μg/kg 240 q3dX5 9.5 deJ591 12.96 mg/kg 0 q3dX5 1.9 deJ591-DMl 4.8 mg/kg90 q3dX5 31.9 deJ591-DMl 6.5 mg/kg 120 q3dX5 37.8 deJ591-DM1 9.7 mg/kg180 q3dX5 42.7 deJ591-DMl 12.93 mg/kg 240 q3dX5 46.4 deJ591-DM1 6.5mg/kg 120 q7dX5 33.9 deJ591-DM1 12.93 mg/kg 240 q7dX5 66.8 ^(a)Tumorgrowth delay is the difference in time (days) for the treatment group toreach 1000 mm³ compared with the vehicle-treated group, calculated fromthe mean values.

Results: After the tumor growth delay (TGD) conferred by the initialcourse of deJ591-DM1 treatment at the highest dosage (12.93 mg/kg or 240μg/kg DM1-equivalents) for either the q3dX5 or q7dX5 schedule, groupswere allowed to attain mean tumor volumes of approximately 1000 mm³. Asecond course of treatment was then initiated. Both treatment groupsresponded to the second course of treatment, although the duration ofthe second TGD response was approximately 75 to 80% of the TGD responseseen after the initial treatment. deJ591-DM1 produced potent regressionsin large tumors, ranging in size from 500 to 2000 mm³ in both of thesetreatment groups, demonstrating the efficacy of deJ591-DM1 against bulkyas well as smaller CWR22 prostate cancer xenografts. Finally, serumconcentrations of PSA in deJ591-DM1 treated and control groups weredirectly correlated with tumor volume and response to deJ591-DM1 (seeFIG. 17).

In conclusion, deJ591-DM1 produced greater tumor growth delay, ascompared to its constituents (deJ591 and DM1) at equimolar dosages, in adosage- and schedule-dependent manner. Additionally, the CWR22 xenografttumor responded to a second course of deJ591-DM1 after outgrowthfollowing response to the initial course. Tumor growth and response todeJ591-DM1 was directly correlated with serum PSA concentration.

deJ591-DM1 Efficacy in PSMA-Positive CWR22 Xenografts: Dosing IntervalComparison of 7, 14, 21, and 28 Days

Previous studies of deJ591-DM1 efficacy in the CWR22 xenograft modelusing C.B-17 scid mice had found an increased TGD with increased dosinginterval at the maximum dosage given (240 μg/kg DM1-equivalents). Theobjective of this study was to determine how much the dosing intervalcould be increased at this dosage before a loss of efficacy in terms ofTGD would be observed. Dosing intervals of 7, 14, 21, and 28 days at adosage of 12.93 mg/kg deJ591-DM1 (representing 240 μg/kgDM1-equivalents) were tested for five cycles. As is shown in FIG. 18,the schedule of q14d was found to be optimal in this model, yielding aTGD for the CWR22 xenograft of 75.7 days. The growth delays for theother schedules were: q7d, 53.0 days; q21d, 48.0 days; and q28d, 26.9days. Similar to other studies using the CWR22 xenograft model, nocurative responses were observed, with all tumors eventually resuminggrowth (FIG. 18). The schedule using a 14-day interval at this dosage(240 μg/kg DM1-equivalents) may be optimal for CWR22 xenograft TGD andsuggests tumor burden can be controlled in this model with continuedtreatment.

deJ591-DM1 Efficacy in Castrate or Intact Scid Mice Bearing theAndrogen-Independent PSMA-Positive 22RV1 Xenograft

Prostate tumors can be broadly classified as either androgen-dependentor androgen-independent. PSMA expression has been suggested to beinversely influenced by androgens. The objective of this study was toevaluate deJ591-DM1 in a model of prostate tumor growth where androgenlevels have been reduced. The 22RV1 cell line was originally developedas androgen-independent and can be grown as a xenograft in C.B-17 scidmice that have been left either intact or castrated. In mice castrated10 days prior to inoculation with the 22RV1 cells, the tumors reached1000 mm³ in 19.6 days. This was approximately twice the time requiredfor the tumors of intact mice to reach 1000 mm³ (9.3 days). C.B-17 scidmice bearing CWR22 xenografts approximately 200 mm³ in size received IVinjections (200 μL constant volume) of the test articles according tothe dosage and schedule shown in the Table 24.

TABLE 24 Dosage, Schedule, and Response of Castrate or Androgen-IntactScid Mice Bearing PSMA-positive 22RV1 Xenografts Tumor DM1- Growth Testequivalents Delay^(a) Relative Article Dosage (μg/kg) Schedule (Days)TGD^(b) Intact Mice Vehicle 0 0 q3dX5 0 NA_(C) DM1 240 μg/kg 240 q3dX56.3 NA MLN2704 12.93 mg/kg 240 q3dX5 13.1 NA MLN2704 12.93 mg/kg 240q7dX5 7.8 NA Castrate Mice Vehicle 0 0 q3dX5 0 NA DM1 240 μg/kg 240q3dX5 9.1 1.4 MLN2704 12.93 mg/kg 240 q3dX5 26.3 2.0 MLN2704 12.93 mg/kg240 q7dX5 21.4 2.7 ^(a)Tumor growth delay is the difference in time(days) for the treatment group to reach 1000 mm₃ compared with thevehicle-treated group, calculated from the mean values. ^(b)Relative TGD= TGD of castrate mice/TGD of intact mice. _(C)Not applicable.

Results: For the tumor model with the androgen-intact mice, deJ591-DM1produced a better TGD on the q3d schedule when compared to DM1 given atequivalent dose and schedule. The response to deJ591-DM1 given on a q7dschedule in the intact mice was not greatly different from that for micetreated with DM1 alone. In the androgen-depleted model, deJ591-DM1showed a benefit at both schedules in terms of TGD, as compared to DM1given at the equivalent dosage in the q3d schedule. Additionally, theTGD values indicate there may be a greater advantage of deJ591-DM1 inthe castrate versus the androgen-intact group, independent of the growthkinetics. The effect of the respective treatments on the change ingrowth delay for castrate mice relative to androgen-intact mice for DM1q3dx5 was 1.4-fold, for deJ591-DM1 given q7dx5 it was 2.7-fold, and fordeJ591-DM1 given q3dx5 there was a 2-fold increase in relative growthdelay. Thus, deJ591-DM1 provides a therapeutic advantage in this modelof tumor growth where androgen levels have been reduced.

In conclusion, in a model of androgen depletion, deJ591-DM1 wasdemonstrated to produce an efficacious response to inhibitandrogen-independent prostate tumor growth.

Efficacy of deJ591-DM1 Compared to the Unconjugated Antibody (deJ591) orthe Unconjugated Tumor Inhibitory Agent (DM1)

Experiment: Male SCID mice were implanted by serial passage of CWR22prostate tumor xenograft. When these tumors reached 200-250 mm³ size(estimated from external caliper measurement), mice were randomized intotreatment groups of 8 to receive vehicle only, deJ591-DM1 at a dose of14.5 mg/kg antibody conjugate (equivalent to 240 ug/kg DM1), deJ591 atthe same dose as deJ591-DM1, or DM1 given at a dose of 240 ug/kg. Alltreatments were given intravenously and on a schedule of every threedays for 5 doses. Tumor growth and animal health were continuallymonitored throughout the study with tumor growth measured every 3 days.

Results: The unconjugated anti-PSMA antibody (deJ591) had no significanteffect on reduction in the rate or extent of CWR22 xenograft tumorgrowth. DM1 administered as free drug produced some tumor growth delay,but was a minor response compared to the deJ591-DM1 administered on thesame schedule with the same molar equivalent of the active DM1. ThedeJ591-DM1 produced a suppression of tumor growth for approximately 20days following the last administered dose.

Efficacy of DEJ591-DM1 at Different Doses

Experiment: Male SCID mice were implanted by serial passage of CWR22prostate tumor xenograft. When these tumors reached 200-250 mm³ size(estimated from external caliper measurement), mice were randomized intotreatment groups of 8 to receive vehicle only, deJ591-DM1 at a dose of14.5 mg/kg antibody conjugate (equivalent to 240 ug/kg DM1), or a lowerdose of 7.25 mg/kg. All treatments were given intravenously and on aschedule of every seven days for 5 doses. Tumor growth and animal healthwere continually monitored throughout the study with tumor growthmeasured every 3 days.

Results: A dose response relationship is evident for the CWR22 xenografttumor growth inhibition by deJ591-DM1. This is shown on a 7 day dosinginterval study for 2 different doses of deJ591-DM1. At the higher dose,there is suppression of tumor growth with some reduction from theinitial tumor volume. At the lower dose there is not the same reductionfrom initial tumor volume and there is a more rapid return to normalgrowth kinetics of approximately 10 days following the last doseadministered compared to the higher dose.

Example 20 Bone Marrow Involvement in Advanced Prostate Cancer PatientsDemonstrated on Bone Marrow Biopsy

Bone marrow involvement in advanced prostate cancer is not routinelyexamined.

We report on the results of bone marrow biopsies performed on advanced,hormone-refractory prostate cancer patients.

Screening diagnostic studies were performed on hormone-refractoryprostate cancer patients to determine eligibility for two phase Iradioimmunotherapy clinical trials. Studies included serologic testing,bone scan, CT scans of the head, chest, abdomen and pelvis, and a bonemarrow biopsy taken from the iliac crest. A total of Thirty-ninepatients have been screened thus far.

All patients had advanced disease as determined by bony or soft tissuemetastases on imaging and/or three consecutive rises in serum PSAlevels. All patients had received prior hormonal therapy, and themajority had received local therapy, including radical prostatectomy(N=15), radiotherapy (N=19), and/or chemotherapy (N=19). Sixteenpatients (41%) had histologic evidence of metastatic prostate cancer onbone marrow biopsy. Of the thirty-nine screened patients, thirteen (33%)had significant bone marrow involvement (>10% involvement), making themineligible for entry into these clinical trials.

Patients with bone marrow involvement had significantly higher serumalkaline phosphatase (ALP) levels (median 374 U/L vs. 96 U/L, p<0.001)and significantly lower serum hemoglobin (median 11.6 g/dL vs. 12.7g/dL, p=0.02). There was no difference in serum hemoglobin betweenpatients with prior chemotherapy and/or prior radiotherapy. When the ALPwas normal (<120 U/L) or elevated (>120 U/L,) 0% and 75% of patients,respectively, had metastatic prostate cancer on bone marrow biopsy(p<0.0001). Patients with bone scans indicating bony metastases in threeor more different anatomic sites (spine, thorax, pelvis, appendicular orcalvarium) vs. two or less sites, were more likely to have bone marrowinvolvement (54% vs. 9%, p=0.01). Age, initial Gleason sum, serum PSA,PSA doubling time, the presence of soft-tissue metastases, prior localtreatment or chemotherapy, or other hematologic parameters (WBC,platelet count) were not significantly different between the two groups.

Bone marrow involvement in advanced prostate cancer patients is notroutinely examined, but is present in a large minority of cases. Currentclinical staging studies may significantly underestimate bone marrowinvolvement, although elevation of alkaline phosphatase or depressedserum hemoglobin may be correlative. This situation should be consideredin clinical trials carrying potential hematologic toxicity.

Example 21 A Novel Sandwich Enzyme-Linked Immunoassay (ELISA) forQuantification of Prostate-Specific Membrane Antigen

Ideally, serum PSMA should be detectable with a simple, rapid,reproducible and quantitative ELISA assay. Our objective was toestablish an ELISA assay to measure serum PSMA.

Ninety-six well plates were coated with an anti-PSMA antibody as a“capture” antibody. Dilutions of serum from males and females “spiked”with recombinant PSMA (rPSMA), semen, LNCaP lysates, as well as thestandard (rPSMA range 1.6-1600 ng/ml) were then added to these wells. Anon-competing biotinylated anti-PSMA antibody (that recognizes adifferent epitope on PSMA) was then added as the “detection” antibody.Avidin phosphatase followed by p-nitrophenyl phosphate (substrate) wasadded. Optical densities were then measured.

The standard curve for the assay was linear through a range of 5-1600ng/ml (correlation coefficient>0.99). Using this assay, PSMA wasdetected in LNCaP lysate, semen, and “spiked” serum.

Example 22 deJ591-DM1 Reference Standard

Table 25 lists the analytical values for a reference batch ofdeJ591-DM1.

TABLE 25 deJ591 Reference Standards Batch Number 1552-60S N067020302Test Result Result Appearance Clear, colorless solution, Clear,colorless; white particle free particulates pH 5.6 5.5 MLN2704Concentration by UV 2.5 mg/mL 4.7 mg/mL [mg/mL] DM1/MLN591 ratio DM1 3.73.5 determined by UV Potency: in vitro cytotoxicity Reference 94%towards PSMA-positive cells [% of reference] Size exclusionchromatography 97% 98% % Monomer Molecular Integrity Reference Bandingpattern conforms to Reduced SDS-PAGE reference % H + L 92% 96% MolecularIntegrity Reference Banding pattern conforms to Non-reduced SDS-PAGEreference; Isoelectric focusing Reference Banding pattern conforms toreference Acetone extractable DM1 3% 2% [% of total DM1] Endotoxin[EU/mg] <0.09 EU/mg <0.04 EU/mg Bioburden [CFU/mL] <1 CFU/mL <1 CFU/mLDM1-related impurities [RP- + HPLC] DMl-thiopenlanoic acid 1.73 μM [2.1%of total 3.07 μM [2.8% of total DM1] DM1] DM1 dimer Not detected 0.43 μM[0.39% of total DM1] DM1 Not detected 0.98 μM [0.88% of total DM1]Molecular Weight [MALDI] 150,237 150,409 ECSO binding to LNCaP [FACS]7.9 nM 6.9 nM Relative binding [% of reference] Reference 114% Sizeexclusion chromatography % 2.8% 2.5% Aggregate + Dimer Binding constantfor binding to 5 × 10⁻⁹ M⁻¹ 7 × 10⁻⁹ M⁻¹ recombinant PSMA [BiaCore]Molecular Integrity Non-reduced SDS-PAGE % IgG {140-160 kDa bands] 89%92% Unconjugated antibody [IEF] <2% <2% Unconjugated antibody 1.0% 1.2%[depletion ELISA] Non-specific in vitro cytotoxicity towardsPSMA-negative cells ICSO [nM] 13.6 nM 17.1 nM In vivo activity - mousexenograft Delays tumor growth Not yet tested model

Example 23 Quantifying DOTA-NHS in Solution

Due to the unstable nature of DOTA-NHS in solution, the quality (e.g.,concentration) of the DOTA-NHS starting material must be controlled inorder to obtain consistent DM1/deJ591 conjugation ratios. Thus, a methodfor indirectly quantifying DOTA-NHS is provided.

The DOTA-NHS hydrolysis reaction is a first order reaction because H₂Ois in large excess. At time 0, the DOTA-NHS concentration is C₀. At timet, the DOTA-NHS concentration is C_(t). From the first order reaction:

${{- \frac{\lbrack C\rbrack}{t}} = {k\lbrack C\rbrack}};{{- \frac{\lbrack C\rbrack}{\lbrack C\rbrack}} = {k{t}}};{{- {\int\frac{\lbrack C\rbrack}{\lbrack C\rbrack}}} = {\int{k{t}}}}$ln [C₀^(′)] − ln [C_(t)^(′)] = kt

Since the conversion ratio between DOTA-NHS and DOTA is 1:1,empirircally determined DOTA concentration can be used to replace [Co₁]and [C_(t1)]. C_(max) is the concentration of DOTA after the hydrolysisreaction is complete. C₀ is the concentration of DOTA at time 0. C_(t)is the concentration of DOTA at time t.

C ₀ ^(t) =C _(max) −C ₀ ; C _(t) ¹ =C _(max) −C _(t);

ln[C _(max) −C _(t) ]=−kt+ln[C _(max) −C ₀]  (2)

C_(max) and C_(t) are determined through a time course experiment, andln[C_(max)−C_(t)] is plotted to give a linear line. The intercept of Yis ln[C_(max)−C₀], thus the concentration [C₀₁] (μM) of DOTA-NHS can beobtained by linear regression. The concentration unit can be convertedto ng/ml by multiplying [Co₁] (μM) by 501 g/mol.

Methods:

Chemicals. Trifluoroacetic acid (TFA), water and methanol were obtainedfrom J. T. Baker (Phillipsburg, N.J. 08865). DOTA was from StremChemicals (Newburyport, Mass. 01950). N-hydroxysuccinimide (NETS) waspurchased from Pierce (Rockford, Ill. 61105). DOTA-NHS.PF6 was obtainedfrom Macrocyclics (Dallas, Tex. 75252). Sinapic acid for use as a MALDImatrix for DOTA-deJ591 determination was purchased from Fluka(Milwaukee, Wis. 53201). Ethylenediaminetetraacetic Acid (EDTA) was fromSigma (St. Louis, Mo. 631781). ICP grade nitric acid was purchased fromsigma. Distilled water was passed thru a Milli-Q Element A10 System(Millipore). A standard mix of 1 mg/mL iron, nickel, cobalt, copper,zinc, lanthanum, cerium, and lead was custom made by Inorganic Ventures(Lakewood, N.J. 08701).

Liquid Chromatography. A binary high-pressure mixing pump (1100 series,Agilent, Palo Alto, Calif. 94303) was used to perform chromatographicseparation. The pump was operated at 0.5 ml/min during sample analysis.A Luna CN 30×4.6 mm 3 μm (Phenomenex, Torrance, Calif. 90501) was usedas the separation column. Mobile phase A was 0.1% TFA in water, andmobile phase B was 0.1% TFA in 90% MeOH. The LC run was performed atisocratic of 10% of mobile phase B, 90% of mobile phase A for 2.5minutes. A Gilson 235 autosampler (Gilson, Middleton, Wis. 53562) wasused in this work with an injection volume of 10 μL. Before and aftereach injection, the inside probe, outside probe and injection valve wereflushed with 500 μL of water.

Mass Spectrometer. A triple quadrupole mass spectrometer (APT 3000,Sciex, ON, Canada L4K 4V8) with turbo ion spray ionization source wasused as the detector. The positive ion mode was used for DOTA andDOTA-NHS detection. The negative ion mode was used for NHS detection.The electrospray voltage was maintained at 5000V for positive ion modeand −4500V for negative ion mode. The mass spectrometer was operated inmultiple reaction monitoring (MRM) with unit mass resolution for bothmass analyzers. The following transitions were monitored: DOTA, m/z405.4>>203.3; DOTA-NHS, m/z 502>>03.2; NHS m/z 114>>1. A dwell time of500 ms was used for all studies. Nitrogen was used as nebulizer (setting8), curtain (setting 8) and collision (setting 4) gas, and was obtainedfrom a nitrogen dewar (235 psi, BOC Gases, Murray Hill, N.J. 07974) withthe gas regulator maintained at 90 psi for nebulizer gas (gas 1) andturbo gas (gas 2), with another regulator maintained at 50 psi forcurtain gas. The turbo ion spray LC/MS interface was maintained at 450°C. with a nitrogen gas flow of 6.5 L/min.

Standards and QC Solutions. Two separate weighings of DOTA were doneaccurately to the second decimal point of mg level (around 2 mg); onewas used for standard calibration curve preparation, and the other wasused for quality control (QC) solution preparation. Both samples weredissolved in 1 ml of water to be used as stock solutions. The calculatedamount of stock solution was added into 10 ml of water to obtain a highstandard and high QC at 8000 ng/ml. A series of volumetric dilutions of8000 ng/ml with water was performed to obtain concentrations of 4000ng/ml, 2000 ng/ml, 1000 ng/ml, 500 ng/ml, 250 ng/ml, 125 ng/ml, 50ng/ml, 25 ng/ml and 10 ng/ml. All the concentrations were used for thestandard curve. Only the 10 ng/ml, 25 ng/ml, 1000 ng/ml and 8000 ng/mlsolutions were used for QC. Saturated EDTA (acid form) solution wasprepared in water, and equal volumes (100 μL) of saturated EDTA solutionand each concentration level of standard solutions and QC solutions weremixed thoroughly in each well of a 96-well plate. The calibrationsolutions thus ranged from 5 ng/ml to 4000 ng/ml with standards of 5,12.5, 25, 62.5, 125, 250, 500, 1000, 2000 and 4000 ng/ml. The QCs wereat the concentration levels of 5, 12.5, 500 and 4000 ng/ml.

Time Course Experiment for the Hydrolysis of DOTA-NHS. About 2 mg (W1)of DOTA-NHS was accurately weighed and dissolved in 1 mL of dryacetonitrile (ACN) as stock solution, as DOTA-NHS is stable in dry ACN.After all the samples for the standard calibration curve and QC had beentested, 20 μL of stock solution of DOTA-NHS was pipetted into 10 mL ofwater and vortexed. The concentration was C_(total) (=2000*W1 ng/ml). At5 minutes, 15 minute, 30 minutes and every 30 minutes afterwards untilthe signal of DOTA plateaued, the concentration of DOTA was checked byremoving 100 μL of solution and mixing it with 100 μL of saturated EDTAsolution. The concentration of DOTA in ng/mL at each time point wasobtained by 2 times the results from calibration curve. Units of ng/mLwere converted to μM by dividing the concentrations for each time pointby 404 g/mol (the molecular weight of DOTA). The concentration (μM) ofDOTA produced by the hydrolysis of DOTA-NHS was obtained using equation(2), and units of ng/mL were converted to μM by dividing theconcentrations for each time point by 404 g/mol. The percentage ofDOTA-NHS was obtained by dividing the concentration of DOTA-NHS in ng/mlby the C_(total).

Method Validation. A complete method validation was performed for threeruns of three consecutive days in terms of method specificity, limit ofquantification, precision, accuracy, linearity and range, carryover. Thespecial requirement and maintenance for the whole system was alsoexploded. Each run contained duplicate calibration curve standards at 10concentrations and QC samples at 4 concentrations (n=6 at eachconcentration, including at the LLOQ). Each run also included a blankfollowing the highest concentration of standard to evaluate thecarryover of the method. The minimum acceptance criteria for thevalidation are described below:

-   -   (a) Specificity. As DOTA-NHS and NHS may interfere with the        detection of DOTA, acceptable selectivity was defined as no        interference with the detection of DOTA from both DOTA-NHS and        NHS.    -   (b) Precision and accuracy. The precision and accuracy of the        method were validated based on six replicates of high QC (4000        ng/ml), middle QC (500 ng/ml), low QC (12.5 ng/ml) and lower        limit of quantification (LLOQ, 5 ng/ml) concentrations. The mean        value should be within 15% of the actual value except at LLOQ        where it should not deviate by more than 20%. The precision        determined at each concentration level should not exceed 15% of        the coefficient of variation (CV) except for the LLOQ where it        should not exceed 20% of the CV.    -   (c) Lower Limit of Quantification (LLOQ). LLOQ is the lowest        standard on the calibration curve that can be measured with        acceptable precision (20%) and accuracy (80-120%). The analyte        response at the LLOQ should be at least 5 times the response        compared to blank response; normally the signal to noise ratio        from the analyte response is 10. The LLOQ for this method was        determined to be 5 ng/ml.    -   (d) Linearity and range. The calibration range of DOTA was from        5 ng/ml to 4000 ng/ml with standard concentrations at 5, 12.5,        25, 62.5, 125, 250, 500, 1000, 2000 and 4000 ng/ml. Duplicate        injections of each standard solution were performed. The        correlation coefficient of the calibration curve should be        better than 0.95. The deviation of calibration standards from        nominal concentration should be less than 15%, though a 20% or        less deviation from nominal concentration is acceptable for        LLOQ.    -   (e) Carryover. The carryover of the method was checked by        injection of blank (water) followed by injection of the highest        concentration standard (4000 ng/ml). The carryover should be        lower than 20% of signal level of LLOQ.

MALDI TOF for DOTA-J591 Molecular Weight Determination. A MALDI TOFinstrument (Voyager Elite, Applied Biosystem, Framingham, Mass. 01701)was used to determine how many DOTA molecules conjugated to each deJ591molecule before and after controlling the quality of the DOTA-NHSstarting material. Sinapic acid at 10 mg/ml in 50/50 ACN/H₂O with 0.1%TFA was used as the MALDI matrix. The naked deJ591 and deJ591-DOTAcomplex were provided in solution of 0.3M ammonium acetate buffer, pH6.8, at an antibody concentration of approximately 5 to 10 mg/ml. ThedeJ591-DOTA was desalted using G25 UltraMicroSpin column (Nest Group,Inc.) by first adding 200 ul of milliQ water to the column andcentrifuging at 5000 rpm for 3 min, then transferring the column to anew collection tube, loading 25 ul of deJ591-DOTA-containing solution tothe bed of column, and centrifuging at 5000 rpm for 3 min. The resultingpurified sample was eluted into a collection tube; the concentration ofthis sample was then adjusted to approximately 0.5 to 1.0 mg/ml ofantibody by the addition of 0.1% trifluoroacetic acid in milliQ water.The volume ratio of 1:1 for samples to matrix was used and 1 μL ofmixture was deposited to the 10×10 MALDI plate. A nitrogen laseroperated at 4 HZ at 337 nm was used for the MALD1 experiments, with alaser spot size of around 100 μm. Each spectrum was the accumulation of200 laser shots. The MALDI TOF detection mode was positive and linearwith delayed extraction.

ICPMS. Measurements for ICPMS were taken on a Platform ICP (Micromass,UK). The Platform ICP is a collision/reaction cell based instrument thateliminates argon based interferences with a mixture of helium andhydrogen. These interference peaks are numerous at the lower end of thespectrum and preclude measurement in this region if not eliminated.

ICPMS operating conditions: cooling gas 13.00 L/min, plasma power xxxxW,plasma gas×L/min acquisition mode SIR, nebulizer gas 0.8 L/min, dwelltime 0.2 s, helium gas 4.0 mL/min, hydrogen gas 3.5 mL/min. Samples wereintroduced into the plasma with a Meinhard nebulizer.

Multielement analysis. The DOTA-NHS lots were diluted in 1% nitric acidand infused onto the ICPMS by passing through an ARIDUS desolvator. Afull scan mode was used with the range being 5 to 250 amu. Theintensities for 56 Fe, 58 Ni, 59 Co, 63 Cu, 64 Zn, 139 La, 140 Ce, and208 Pb were compared to a 10 ppb standard of Co. The nitric acid diluentwas used for background subtraction. Using elemental targeting with theData Explorer software (ABI), matches were made for the isotope patternsfor each of the desired elements. DOTA-NHS and deJ591-DOTA conjugatesamples were diluted in 1% nitric acid to a concentration of 100 mg/mL.Samples were run in both SIR and full scan mode. A full scan mode wasused with the range being 5 to 250 amu. Masses monitored for selectedion recording (SIR) mode were 56 Fe, 58 Ni, 59 Co, 63 Cu, 64 Zn, 139 La,140 Ce, and 208 Pb. Calibration curves for the standard mix weregenerated simultaneously for 100, 10, 1, 0.1, 0.01, and 0.001 ppb. usingMasslynx software.

Results:

FIG. 19 shows the overlay of naked deJ591 and two batches of DOTA-NHSand deJ591 conjugation results. The left-most trace is the 2+ chargestate of naked deJ591; the average mass is 73740. The average mass forone batch of conjugation experiment (2+ charge state of DOTA-J591conjugates, middle trace) is 74707 and for the other batch (2+ chargestate of DOTA-J591 conjugate, right-most trace) is 75462. The 2+ chargestate (approx. 74 kDa) is chosen for mass shift calculation over the 1+charge state peak since mass accuracy and resolution is better at the 2+mass compared to 1+ charge state peak (approx 148 kDa). Resolution for2+ charge state peak is 60-80 at fwhm (full width half maximum)definition for naked deJ591 2+ charge state peak compared to resolutionof 30-40 fwhm for DOTA conjugated deJ591 2+ charge state peak. Thecentroid mass of 2+ charge is converted to zero charge mass bymultiplying the observed mass by a factor of 2 then subtracting 2(1.0079 mass of hydrogen) mass units. The number of DOTA moleculesconjugated to each antibody molecule equals the mass difference betweenthe calculated zero charge state mass peak centroids for naked deJ591and DOTA-J591, divided by 386 (386 is the mass of the addition of amolecule of DOTA when conjugated to Lysine). The calculated conjugationratio between DOTA and deJ591 is 8.7 and 4.8 respectively, for theright-most trace and middle trace. As one theory, the variation inconjugation ratio is due to a lack of quality control of the DOTA-NHSstarting material since DOTA-NHS is not stable.

The availability of DOTA-NHS in different batches can be determined byquantifying DOTA at different time points during a real time kinetics ofhydrolysis experiment. FIG. 20A shows an example of real time kineticsof DOTA-NHS hydrolysis experiment. According to formula (2), plotln[C_(max)−Co] versus time t, a linear line has been obtained (FIG.20B). The correlation coefficient is 0.9979, which proves that DOTA-NHShydrolysis is a first order reaction. The intercept of Y isln[C_(max)−Co], so the concentration (Co′) of DOTA-NHS which correspondsto (C_(max)−C₀) can be obtained.

FIG. 21 shows the MALDI MS spectra data for different conjugationbatches between DOTA-NHS and deJ591 after the calculation ofavailability of DOTA-NHS in different batches just before theconjugation experiments to control the quality of the starting material.The real input of molar ratio of DOTA-NHS to deJ591 is now keptconstant, and as a result, the conjugation ratio of DOTA to deJ591becomes consistent from batch to batch. To further examine the actuallevel of DOTA conjugation ratios to the deJ591 antibody, thedistribution ratio of DOTA conjugation to the deJ591 antibody can beinvestigated using Gaussian Deconvolution and Peak Fitting software(PeakFit, Systat, Inc.); processing the peak data from MALDI-TOF MSresults for 2+ charge state peaks. Since the DOTA conjugated deJ591 peakshown in FIG. 21 exhibits much lower resolution compared to the nakeddeJ591 peak, with peak width approximately double the width of nakeddeJ591 peak, it can be assumed that the reason for this is the DOTAconjugation to deJ591 results in a ratio of conjugation levels,resulting in a heterogeneous non-resolved peak representing adistribution of various DOTA conjugation levels.

FIG. 22 represents the data from FIG. 21 after subjecting the 2+ chargestate peak to first Gaussian deconvolution followed by Gaussian peakfitting. Comparing this processed peak data to the naked deJ591 centroidmeasurement provides the ability to identify the resolved and fittedpeak for zero conjugation, and the adjacent peaks representing variouslevels of DOTA conjugation resulting from mass differences betweenresolved and fitted peaks across the 2+ charge state peak signal (FIG.23) display DOTA conjugation levels from zero DOTA up to 7 DOTA. Theaverage conjugation level, based on total peak centroid measurement(FIG. 21) of 5.7 DOTA. The resulting mass differences between the fittedpeaks is an average of 518 with a % CV of 3.2%; while the expected massdifference for each DOTA conjugated is a mass addition of 386, andprevious measurements of DOTA-peptide conjugates using monoisotopicresolved mass assignment has confirmed this. As one theory, not meant tobe limiting, the higher values observed for each DOTA conjugation to theantibody could be from the Gaussian deconvolution and peak fittingprocess, having only minimal representation of these DOTA conjugationpeaks present in the raw data, then extrapolating these with softwareprocessing; another non-limiting possibility is the presence of anunidentified contaminant forming an adduct ion with DOTA conjugatedantibody or DOTA molecules, when this intact antibody is analyzed byMALDI-TOF MS.

Example 24 Pharmacokinetics of deJ591 and deJ591-DM1 in Mice andCynomolgus Monkeys

The objectives of this GLP-compliant study were to determine thepharmacokinetic properties of deJ591 and deJ591-DM1 and to evaluate thepotential immunogenicity of deJ591 and deJ591-DM1.

Briefly, male CD-1 mice received a single IV injection via tail vein of10, 30 or 90 mg deJ591-DM1/kg. Blood was collected from 3 mice/timepoint at specified time points (0 [non-dosed], 5, 15, and 30 minutes and1, 2, 4, 8, and 24 hours after dosing and on Days 3, 5, 8, and 15) fordetermination of deJ591-DM1 and total deJ591 serum concentrations andsubsequent pharmacokinetic analysis.

Groups of male and female cynomolgus monkeys received saline, deJ591, ordeJ591-DM1. Blood was collected at specified time points fordetermination of serum deJ591-DM1 and total deJ59I concentrations andsubsequent PK analysis. deJ591-DM1 concentrations were determined onlyfor deJ591-DM1-dosed animals, while total deJ591 concentrations weredetermined for both deJ591-DM1- and deJ591-dosed animals. Blood was alsocollected (prior to infusion [0] and on Days 8, 15, 22, and 29) fordetermination of primate anti-MLN591 antibody titers to assessimmunogenicity.

After administration of deJ591-DM1, there was a tendency for the serumconcentrations of deJ591-DM1 to be slightly higher than the serumconcentrations of total deJ591 at early time points. The reason for thisdifference is not presently known. This effect was more pronounced inmonkeys than in mice. Both the deJ591-DM1 and total deJ591 were clearedslowly, with the t_(1/2) for deJ591-DM1 and total deJ591 in mice beingapproximately 21 hours and 46 hours, respectively. The clearance in themonkeys was slower than in mice, with the t₁₁₂ for deJ591-DM1 and totaldeJ591 being approximately 54 and 198 hours, respectively. The half-lifewas dosage-independent, while the exposure (as measured by eitherC_(max) or AUC) increased as the dosage increased, with the increasebeing approximately dosage proportional. The exposure to total deJ591was 2- to 4-fold greater than the exposure to deJ591-DM1. Thedosage-normalized exposure to deJ591-DM1 tended to be higher in monkeysthan in mice when the dosage was expressed on an mg/kg basis. However,when the dosage was expressed on an mg/m² basis, the dosage-normalizedexposures to deJ591-DM1 in mice and monkeys were similar. As one theory,not meant to be limiting, it is postulated that the faster clearance ofdeJ591-DM1 is due to deconjugation of deJ591-DM1 in the RES followingcellular processing of the immunoglobulin to generate deJ591 and DM1rather than due to a more rapid clearance of the immunoconjugate bynormal antibody clearance mechanisms. The PK parameters of deJ591-DM1and total deJ591 in mice and monkeys are summarized in Table 26.

TABLE 26 Summary of the PK Parameters of deJ591-DM1 and Total deJ591 inCD-1 Mice and Cynomolgus Monkeys deJ591-DMl Total deJ591 Test StudyDosage Dosage C_(max) T_(max) ^(a) AUC^(b) t_(1/2) C_(max) T_(max)AUC^(b) t_(1/2) Species Gender Article Number (mg/kg) (mg/m²) (μg/mL)(h) (μg*h/mL) (h) (μg/mL) (h) (μg*h/mL) (h) Mouse Male deJ591- CTBR 1030 196 0.5  3260 21.5 199 0.5  8920 52.3 DMl 57842 30 90 459  0.08  596022.5 471  0.08 21000 46.0 90 270 2110  0.08 30600 20.1 2020   0.08 6230039.2 Mean  NA^(d)  0.22 NA 21.4 NA  0.22 NA 45.8 (SD)_(C)  (0.24) (1.21)  (0.24) (6.55) Monkey Male & deJ591- KLA 6 72 157 1    5020 45  178 5   19900 133 Female DM1 W-171 (29.8) (0.6)  (757)  (5.7)   (49.5)(3.5)  (7920) (21.0) Monkey Male & deJ591 6 72 NA NA NA NA 176 1   25200164 Female   (28.1) (0.5)  (5930) (12.9) Monkey Male & deJ591- CTBR 6 72213 0.8  6720 50.9 181 1.0 17200 170 Female DM1 57355 10 120 339 0.610600 51.0 291 1.3 31700 200 16 192 506 0.8 15600 50.4 467 0.9 44400 17130 360 1210 0.8 33500 61.7 1140  1.1 95000 253 Monkey Male & deJ591-CTBR Mean NA NA 0.7 NA 53.5 NA 0.9 NA 198 Female DMl 57355 (SD) (0.4)(11.0) (0.7) (48.5) Monkey Male & deJ591 CTBR 10 120 NA NA NA NA 315 0.649400 251 Female 57355 ^(a)Time after the start of the infusion (monkey)or bolus injection (mouse). ^(b)Mouse AUC = 0-336 hours; Monkey AUC =0.672 hours, _(C)Standard deviation. ^(d)Not applicable.

Example 25 Toxicity of deJ591 and deJ591-DM1 in Mice and CynomolgusMonkeys

DM1 is a structural analogue of maytansine, a naturally occurring,cytotoxic, macrocyclic antibiotic. DM1 blocks the polymerization oftubulin, thus inhibiting microtubular formation and mitosis and inducingmetaphase arrest of dividing cells in vitro. For maytansine, thisstathmokinetic cytotoxicity is irreversible and specific for G2 and Mphase cells, with histologic evidence of arrest in metaphase, suggestingimpaired mitotic spindle formation. DM1, and thus deJ591-DM1, would beexpected to share this mechanism of action and therefore to be aneugenic(capable of causing irregularities in chromosomal numbers), but notclastogenic (capable of causing chromosomal fragmentation) or mutagenic(capable of causing direct damage to DNA). deJ591-DM1 was demonstratedto be positive for the induction of numerical chromosome aberrations(polyploidy/endoreduplication) in CHO cells in vitro at 500 mg/mL,likely related to aneugenicity. In vivo deJ591-DM1 was positive at 30,48, and 90 mg/m² for increases in micronucleated polychromaticerythrocytes in the mouse micronucleus test. deJ591-DM1 was notmutagenic in a bacterial reverse mutation assay (Ames' test).

CD-1 mice and cynomolgus monkeys were chosen as the rodent and nonhumanprimate species for nonclinical toxicology testing of deJ591-DM1 on thebasis of published recommendations for establishing the human SafeStarting Dose (SSD) for Phase 1 clinical trials of immunoconjugates.Cynomolgus monkeys were additionally selected because they sharedsimilar tissue cross-reactivity patterns with human tissues,characterized by strong immunostaining of epididymal epithelium and weakimmunoreactivity in glial cells and neuropil in the subcortical whitematter of brain. However, in contrast to human tissues, monkey prostateepithelial cells did not stain (monkeys do not express PSMA on prostateepithelium). Mice were additionally because they were used in prostatecancer xenograft models to evaluate the pharmacology of deJ591-DM1 andin genotoxicity tests. Use of both mice and cynomolgus monkeys fornonclinical testing allowed comparison of exposure and toxicity acrossspecies on the basis of body surface area (mg/m²), and thus aided inselecting the SSD for the proposed Phase 1 clinical trial.

A single IV administration of deJ591-DM1 induced dosage-dependenttoxicological changes in many organ systems or tissues in mice andmonkeys. Toxicity (in mice) was characterized microscopically as“mitosis/necrosis”, which appeared to represent arrest of the mitoticcycle, with subsequent cell death. This microscopic finding iscompatible with the known mechanism of toxicity of DM1, and was alsoseen after administration of nonconjugated DM1 to mice. In both monkeysand mice, target organs identified for deJ591-DM1 included:gastrointestinal, hematopoietic (bone marrow), liver and lymphoidtissue. No clinical or microscopic evidence of toxicity to neuraltissues was observed in either species. In monkeys dosed withdeJ591-DM1, mild to moderate microscopic changes in the target organswere only seen at the severely toxic dosage (STD) of 360 mg/m², whichresulted in mortality in 25% of the animals. The highest non-severelytoxic dosage (HNSTD) or maximum-tolerated (non-lethal) dosage (MTD) formonkeys was 192 mg/m, where toxicity was limited to clinical signscompatible with mild gastrointestinal toxicity in a few animals.

For mice, the MTD was 180 mg/m², which was slightly less than that formonkeys, yet considerably more toxic. At the MTD for mice moderate tomarked toxicity affected gastrointestinal, hematopoietic (bone marrow),liver, and lymphoid (thymus, spleen, lymph nodes, gut-associatedlymphoid tissue [GALT]) organs, and in addition, reproductive (ovaries,uterus, testes, seminal vesicles, epididymis, prostate) and many otherglandular (mandibular salivary, lacrimal, Harderian, and adrenal glands)organs or epithelial tissues. The calculated dosages that wouldrepresent the LD10 for male and female mice were 289 and 286 mg/m²,respectively. The STD for mice was 300 mg/m2, which was slightly lessthan that for monkeys, though associated with similar mortality (30%).However, at the STD for mice, toxicity occurred in all organs affectedat the MTD and was additionally accompanied by moderate to markedtoxicity of urogenital organs (bladder, vagina, and penis).

The no-observable-adverse-effect level (NOAEL) for deJ591-DM1 in monkeyswas 120 mg/m². In contrast, a NOAEL for dcJ591-DM1 was not identified inmice, although at the lowest tested dosage (30 mg/m²), toxicity waslimited to slightly (10%) lower testes weights. Toxicity to reproductivetissues of male monkeys was not evaluable due to sexual immaturity. Withthe exception of toxicity to testes in mice, most of the changesobserved in both species were partially to completely reversible.

The toxicity of a single IV administration of equimolar dosages of DM1was compared to that for deJ591-DM1 in mice. DM1 tended to be less toxicthan deJ591-DM1 at lower dosages, although the DM1-equivalent MTD andSTD dosages were identical and the DM1-equivalent LD10 dosages for malesand females were similar. Target organs identified for DM1 were similarto those for deJ591-DM1, with a few notable exceptions: liver toxicitywas less prevalent with DM1 and lacked marked elevations in serumtransaminase values, changes to testes weights were seen only at higherdosages, and urogenital organs (including penis) were less affected bytoxicity. The differences in toxicity are considered compatible withdifferential tissue distribution and a greater duration of exposure todeJ591-DM1 than to DM1.

Although guidance documents on selection of the SSD for anticancerimmunoconjugates are not available, published literature suggests theuse of one-tenth the LD10 in mice or one-sixth the HNSTD in nonhumanprimates. Based on the mouse LD10 (289 mg/m²) and monkey HNSTD (192mg/m²), a SSD for deJ591-DM1 of 29 or 32 mg/m², respectively, can besupported. In light of the fact that primates appear more resistant tothe toxicity of deJ591-DM1 than do mice, the proposed SSD for the Phase1 human clinical trial is 32 mg/m².

Results from the toxicological studies are summarized in Tables 27-29below.

TABLE 27 Comparison of Toxicity of deJ591-DMl After a Single IntravenousAdministration to CD-1 Mice or Cynomolgus Monkeys at Various DosagesCD-1 Mice (CTBR 57351) Cynomolgus Monkeys (CTBR 57355) deJ591- deJ591-deJ591- DM1 DM1 DM1 Dosage Dosage Degree of Toxicity and Major TargetDosage Degree of Toxicity and Major Target (mg/m²) (mg/kg) Organs(mg/kg) Organs Comments 30 10 Mild toxicity to testes. Lowest genotoxicdosage in ICR mice 48 16 Mild to moderate toxicity to reproductive(ovaries, uterus, testes) organs. 72 6 No effects. 90 30 Mild tomoderate toxicity to reproductive, gastrointestinal, hepatic, lymphoid,and bone marrow organs. 120 10 No effects. Monkey NOAEL 180 60 Moderateto marked toxicity to Mouse MTD reproductive, gastrointestinal, hepatic,lymphoid, and bone marrow organs. 192 16 Clinical signs compatible withmild Monkey HNSTD gastrointestinal toxicity in a few animals or MTD 28695 Mouse LD₁₀ (female) 289 96 Mouse LD₁₀ (male) 300 100 30% mortalityattributed to severe Mouse STD gastrointestinal and bone marrowtoxicity. Moderate to severe toxicity to reproductive, gastrointestinal,hepatic, lymphoid, bone marrow, and urogenital organs. 360 30 25%mortality attributed to gastrointestinal Monkey STD toxicity. Mild tomoderate, reversible toxicity to gastrointestinal, bone marrow, hepaticand lymphoid tissues. Reproductive toxicity not evaluable in males dueto sexual immaturity. 480 160 100% mortality attributed to severe MouseLD₁₀₀ gastrointestinal and bone marrow toxicity.

TABLE 28 Comparison of Dosages of deJ591-DMl Administered to Animals orProposed for the Phase 1 Clinical Trial and the DMl-equivalents Receivedat Each Dosage CD-1 Mice Cynomolgus Monkeys Humans DM1- DM1- ProposedDM1- deJ591-DM1 deJ591-DM1 Equivalents deJ591-DM1 Equivalents deJ591-DM1Equivalents Dosage Dosage received Dosage received Dosages received(mg/m²) (mg/kg) (mg/m²) (mg/kg) (mg/m²) (mg/kg) (mg/m²) Comments 29 0.80.51 1/10 Male Mouse LD₁₀ 30 10 0.54 Lowest genotoxic dosage in ICR mice32 0.9 0.58 1/6 Monkey HNSTD −42 ~14 ~0.72 Efficacious dosage in SCIDmouse xenografts 48 16 0.86 58 1.6 1.04 72 6 1.30 90 30 1.62 92 2.5 1.66120 10 2.16 Monkey NOAEL 129 3.5 2.32 168 4.5 3.02 180 60 3.24 Mouse MTD192 16 3.60 Monkey HNSTD or MTD 218 5.9 3.92 270 90 4.86 284 7.7 5.11286 95 5.13 Mouse LD₁₀ (female) 289 96 5.18 Mouse LD₁₀ (male) 300 1005.40 Mouse STD 360 30 6.48 Monkey STD 369 10 6.64 480 160 8.64 MouseLD₁₀₀

TABLE 29 Comparison of Toxicity of deJ591-DMl and DM1 after a SingleIntravenous Administration to CD-1 Mice at Equivalent DM1 DosagesMLN2704 in CD-1 Mice (CTBR 57351) DM1 in CD-1 Mice (CTBR 57353) DosageDegree of Toxicity and Dosage Degree of Toxicity and (mg/m²) (mg/kg)Major Target Organs (mg/m²) (mg/kg) Major Target Organs Comments 30 10Mild toxicity to testes. 0.54 0.18 No adverse effect. DM1 NOAEL 48 16Mild to moderate toxicity to 0.86 0.29 Slight mitosis/necrosis ofreproductive (ovaries, uterus, epidermis and adnexa near the testes,epididymis) organs. injection site. 90 30 Mild to moderate toxicity to1.62 0.54 Mild bone marrow myeloid reproductive (ovaries, uterus,hyperplasia, minimal to testes, seminal vesicles, moderate toxicity toepididymis), gastrointestinal, reproductive (prostate, hepatic, lymphoid(thymus, epididymis), gastrointestinal, spleen, lymph nodes, GALT), andand lymphoid (thymus) organs. bone marrow organs. Minimal to slight oroccasionally moderate mitosis/necrosis of liver; slightly- elevated ASTand ALT values. 180 60 Moderate to marked toxicity to 3.24 1.08 Minimalto moderate toxicity to deJ591-DM1 and DM1 reproductive (ovaries,uterus, reproductive (prostate, MTD testes, seminal vesicles,epididymis, testes, seminal epididymis, prostate), vesicles),gastrointestinal gastrointestinal, hepatic, (marked), lymphoid (thymus,lymphoid (thymus, spleen, lymph spleen, lymph nodes, GALT), nodes,GALT), and bone marrow and bone marrow organs. organs. Minimal to slightor Minimal to slight or occasionally occasionally moderate moderatemitosis/necrosis of mitosis/necrosis of liver; liver; no elevated ASTand ALT moderately elevated AST and values. ALT values. 267 89 4.82 1.61DM1 LD₁₀ (males) 270 90 4.86 1.62 286 95 5.13 1.71 deJ591-DMl LD₁₀(females) and DM1 LD₁₀ (females) 289 96 5.18 1.73 deJ591-DMl LD₁₀(males) 300 100 30% mortality attributed to severe 5.40 1.80 80% and 30%mortality in males deJ591-DM1 and DM1 gastrointestinal and bone marrowand females, respectively, STD toxicity. Moderate to severe attributedto severe toxicity to reproductive (ovaries, gastrointestinal and boneuterus, testes, seminal vesicles, marrow toxicity. Marked epididymis,prostate), toxicity to lymphoid (thymus, gastrointestinal, lymphoidspleen, lymph nodes, GALT) in (thymus, spleen, lymph nodes, those dying.Mild to moderate GALT), bone marrow, and toxicity to reproductiveurogenital (bladder, vagina, (prostate, epididymis, testes, penis)organs and various glands seminal vesicles, ovaries, in survivors.Minimal to slight or uterus), lymphoid (thymus only), occasionallymoderate gastrointestinal, bone marrow, mitosis/necrosis of liver; andurogenital (bladder, vagina, markedly elevated AST and ALT but notpenis) organs and values. various glands in those surviving. Minimalhepatic toxicity in one female. Minimal to slight or occasionallymoderate mitosis/necrosis of liver; mildly elevated ALT values (males).480 160 100% mortality attributed to 8.64 2.88 100% mortality attributedto deJ591-DM2 and DM1 severe gastrointestinal and bone severegastrointestinal and bone LD100 marrow toxicity. marrow toxicity.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method of targeting a non-prostate cancer associated withvascular endothelial cells expressing prostate specific membrane antigen(PSMA) in a subject, the method comprising administering to the subjecta first dose of an anti-PSMA antibody, or antigen binding fragmentthereof, the first dose comprising an amount of an anti-PSMA antibody,or antigen binding fragment thereof, effective to target thenon-prostate cancer associated with vascular endothelial cellsexpressing PSMA, wherein the anti-PSMA antibody comprises: a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO:22, the amino acid sequence encoded by the nucleotide sequence shownas nucleotide residues 261-581 of SEQ ID NO:25, or the light chainvariable region amino acid sequence of the antibody produced by the NS0cell line having ATCC Accession Number PTA-3709; and a heavy chainvariable region comprising the amino acid sequence set forth in SEQ IDNO:21, the amino acid sequence encoded by the nucleotide sequence shownas nucleotide residues 261-605 of SEQ ID NO:23, or the heavy chainvariable region amino acid sequence of the antibody produced by the cellline having ATCC Accession Number PTA-3709.
 2. The method of claim 1,wherein the amount of an anti-PSMA antibody, or antigen binding fragmentthereof, effective to target the non-prostate cancer associated withvascular endothelial cells expressing PSMA is selected from the groupconsisting of 5 mg, 10 mg, 20 mg, 40 mg, or 80 mg.
 3. The method ofclaim 1, further comprising administering a second dose of the anti-PSMAantibody, or antigen binding fragment thereof, 14 days after the firstdose of the anti-PSMA antibody, or antigen binding fragment thereof. 4.The method of claim 1, wherein the anti-PSMA antibody comprises twoheavy chains and two light chains.
 5. The method of claim 1, wherein theanti-PSMA antibody, or antigen binding fragment thereof, is conjugatedto a molecular entity.
 6. The method of claim 5, wherein the molecularentity is a therapeutic agent.
 7. The method of claim 6, wherein thetherapeutic agent is a radioactive isotope.
 8. The method of claim 7,wherein the radioactive isotope is ¹¹¹In.
 9. The method of claim 5,wherein the molecular entity is a label.
 10. The method of claim 1,further comprising administering one or more cytotoxic agents.
 11. Themethod of claim 1, further comprising administering one or moreimmunomodulatory agents.
 12. The method of claim 1, wherein the subjectis a human.